Inverse Ugelstad Particles

ABSTRACT

This invention relates to monodisperse cross-linked hydrogel polymer particles comprising a polymer formed from (a) a hydrophilic vinylic monomer; and (b) a crosslinker comprising at least two vinyl groups. The invention also relates to monodisperse seed particles with a Z-average diameter of from 100 nm to 1500 nm that comprise a plurality of non-crosslinked oligomers of poly N,N-dimethylacrylamide. Also provided are methods of forming the monodisperse cross-linked hydrogel polymer particles and monodisperse seed particles.

This invention relates to monodisperse polymer particles useful inbiological assays and other applications, and seed particles used inprocesses of preparing the monodisperse polymer particles. The inventionalso relates to processes for preparing such particles and,intermediates used in such processes and methods of using the particles,as well as other subject matter.

BACKGROUND

Emulsion polymerisation can be used to form relatively monodispersepolymer particles of from 50-1,000 nm. The formation of monodispersepolymer particles does, however, have a number of important limitations.Firstly, all of the particles must be formed as part of the same batchand within a short timespan relative to the polymerization time.Secondly, growth conditions need to be controlled to ensure that growthof the polymer particles is identical in all particles. Thirdly, as theprocess requires that monomer is transferred from large reservoirdroplets and swelled into the growing particles, the particles areeither not crosslinked, or have a very low level of crosslinking, ashigher levels of crosslinking will prevent the particles swelling.

Precipitation or dispersion polymerisation may be used to create largerparticles, of 0.5-10 micron. Precipitation polymerisation is carried outin a solvent (e g alcohols) that dissolves the monomer, but not thepolymer. Monodispersity can be achieved when the polymer precipitatesout of solution and particles formation happens in a short time span. Arelatively large amount of steric stabilizer is required to stabilizethe particles after precipitation. The amount and type of stabilizer maychange the size of the particles, and porous particles can be obtainedby use of a high level of crosslinker. The conditions in precipitationpolymerisation are, however, difficult to control, for example it isvery difficult to control porosity, which is a result of how the porestructure is built up when the polymers precipitate out of solution. Thedifficulties of precipitation polymerisation mean that in practice it isnearly impossible to produce porous particle with the same monomercomposition and porosity for more than a very limited size variation.

One way to address some of the issues is to produce crosslinked porousor solid monodisperse polymer particles by a two stage process, namedthe Ugelstad process after the late Professor John Ugelstad, which isdescribed for example in EP-B-3905 and U.S. Pat. No. 4,530,956. Animproved Ugelstad process is described in WO 00/61647. In the Ugelstadprocess, seed particles, suitably made by emulsion polymerization, areconverted in two steps into monodisperse particles by seeded suspensionpolymerization. In a first step, the seed particles are swollen bymaking a fine (e.g. submicron) aqueous emulsion of a relatively lowmolecular weight water-insoluble substance and then adding awater-miscible organic solvent (e.g. acetone) so that thewater-insoluble substance diffuses into the seed particles. It isconvenient for the water-insoluble substance to be a heat-activatedpolymerisation initiator. In a second step, the solvent is then removed,locking the water-insoluble substance in the seed particles, and theseed particles take up a large quantity of monomer and also acrosslinker. Following initiation of polymerization, e. g. by heating toactivate the initiator, larger polymer particles are produced. TheUgelstad process therefore comprises making seed particles by emulsionpolymerization and expanding the seed particles by suspensionpolymerization. The smallest monodisperse particles described in theaforementioned prior art have an average diameter of 1 μm.

In a simplified version of the Ugelstad process, the enhanced capacityfor swelling may be achieved simply by the use of oligomeric seedparticles, e.g. where the oligomer weight average molecular weightcorresponds to up to 50 monomer units (a molecular weight of about 5000in the case of polystyrene). This is described in U.S. Pat. No.4,530,956. In another version of the Ugelstad process, described inW2010/125170, oligomeric seed particles can be used to make monodisperseparticles with an average diameter in the submicron range.

Particles made by the Ugelstad process and its variants as describedabove are made from hydrophobic monomers, such as styrene, typicallyusing oil (discontinuous phase) in aqueous (continuous phase) systems.The resulting polymer particles are therefore hydrophobic. Hydrophobicparticles suffer from the problem of non-specific absorption when usedin biological applications. This means that hydrophobic polymerparticles are typically surface modified to increase the hydrophilicityof the surface prior to use in biological applications.

US 2014/0073715 describes a method of producing monodisperse hydrophilicparticles. The method uses monomers that have a hydrophobic protectiongroup added to the monomers that polymerize to form the particles andremoving the protection group afterwards. This approach provides goodresults. Addition and removal of the protecting group does, however, addto the complexity of the process and may limit the number of differenttypes of monomer that can be used in the process.

It is apparent that known monodisperse polymeric particles and methodsof making such particles are subject to a number of limitations. Thereis therefore a need for new monodisperse polymeric particles and newmethods of production.

It is an aim of the invention to provide monodisperse polymericparticles and methods of making monodisperse polymeric particles with alow coefficient of variation (CV) and/or low % polydispersity. It isalso an aim of the invention to provide seed particles suitable formaking such monodisperse particles.

BRIEF SUMMARY OF THE DISCLOSURE

The invention is based in part on an appreciation that seed particlescomprising hydrophilic oligomers may be used in a novel process forforming cross-linked monodisperse polymeric particles.

In accordance with a first aspect of the present invention there areprovided monodisperse crosslinked hydrogel polymer particles comprisinga polymer formed from (a) a hydrophilic vinylic monomer having a logP_(oct/wat) (log P) of less than about 1; and (b) a crosslinkercomprising at least two vinyl groups.

A second aspect of the invention provides monodisperse seed particleswith a Z-average diameter of from 100 nm to 1500 nm, wherein each seedparticle comprises a plurality of non-crosslinked oligomers of polyN,N-dimethylacrylamide.

A third aspect the invention provides a method of forming monodisperseseed particles. The method comprises dissolving N,N-dimethylacrylamide,a stabilizer, a radical initiator and a chain transfer agent in anorganic solvent to form a reaction mixture; and heating the reactionmixture to activate the initiator; thereby forming the monodisperse seedparticles.

A fourth aspect comprises the use of seed particles of the invention toform monodisperse crosslinked hydrogel polymer particles. Themonodisperse crosslinked hydrogel polymer particles may be crosslinkedpolyacrylamide particles. The seed particles may be seed particles ofthe second aspect. The seed particles may be seed particles obtainableby or obtained by the method of the third aspect.

A fifth aspect provides a method of forming monodisperse crosslinkedhydrogel polymer particles. The method comprises forming a solution (a)of at least 2% wt of a hydrophilic vinylic monomer in an aqueoussolution, the aqueous solution also comprising a crosslinker comprisingat least two vinyl groups; forming a solution (b) of stabilizer in anorganic solvent, wherein the organic solvent is not miscible in water,and wherein at least one of solution (a) and solution (b) comprises aradical initiator; mixing solutions (a) and (b) to form a water-in-oilemulsion (c) and adding monodisperse seed particles to the emulsion;allowing the monodisperse seed particles to form swollen particles inthe emulsion; and polymerising the swollen particles to form themonodisperse crosslinked hydrogel polymer particles. The seed particlesmay be seed particles of the second aspect. The seed particles may beseed particles obtainable by or obtained by the method of the thirdaspect.

A sixth aspect provides a method of forming monodisperse crosslinkedhydrogel polymer particles. The method comprises forming a solution (a)of at least 2% wt of a hydrophilic vinylic monomer in an aqueoussolution, the aqueous solution also comprising a chain transfer agent;forming a solution (b) of stabilizer in an organic solvent, wherein theorganic solvent is not miscible in water, and wherein at least one ofsolution (a) and solution (b) comprises a radical initiator; mixingsolutions (a) and (b) to form a water-in-oil emulsion (c) and addingmonodisperse seed particles to the emulsion; allowing the monodisperseseed particles to form swollen particles in the emulsion; polymerisingthe swollen particles to form monodisperse polymer particles; forming asolution (d) of stabilizer in an organic solvent, wherein the organicsolvent is not miscible in water; forming a solution (e) of at least 2%wt of a hydrophilic vinylic monomer in an aqueous solution, the aqueoussolution also comprising a crosslinker comprising at least two vinylgroups, wherein at least one of solution (d) and solution (e) comprisesa radical initiator; mixing solutions (d) and (e) to form a water-in-oilemulsion (f) and adding the monodisperse polymer particles to theemulsion; allowing the monodisperse polymer particles to form swollenpolymer particles in the emulsion; and polymerising the swollen polymerparticles to form the monodisperse crosslinked hydrogel polymerparticles. The seed particles may be seed particles of the secondaspect. The seed particles may be seed particles obtainable by orobtained by the method of the third aspect.

A seventh aspect comprises monodisperse crosslinked hydrogel polymerparticles obtainable by the method of the fifth or sixth aspect. Themonodisperse crosslinked hydrogel polymer particles may be obtained bythe method of the fifth or sixth aspect.

An eighth aspect comprises the use of monodisperse crosslinked hydrogelpolymer particles in nucleic acid amplification and/or oligonucleotidesequencing. The nucleic acid amplification may be polymerase chainreaction (PCR) amplification or emulsion PCR amplification. Theoligonucleotide sequencing may be may be chemical field-effecttransistor (chemFET) based sequencing. The oligonucleotide sequencingmay be ion sensitive field-effect transistor (ISFET) based sequencing.

A ninth aspect provides a method for nucleic acid amplificationcomprising conducting a primer extension reaction on a polynucleotidethat is hybridized to an oligonucleotide which is attached to a polymerparticle of the invention.

An embodiment of the invention provides particles which have beenobtained by the processes described in this specification.

An embodiment of the invention provides particles having thecharacteristics of particles obtained by the methods disclosed herein;whilst such particles are obtainable by the processes described herein,they are characterized solely by their properties and not by theirmethod of manufacture and, accordingly, the scope of protection ofclaims directed to particles specified by their characteristics isdetermined solely by the characteristics of the particles to theexclusion of their actual method of manufacture.

The products, processes and uses of the invention are not limited to thesubject matter just-mentioned but are, without limitation, describedmore fully in the following description and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

Embodiments of the invention are further described hereinafter withreference to the accompanying drawings, in which:

FIG. 1 is a diagrammatic representation of a single-stage swellingparticle forming process of the invention, including an indication ofexemplary monomers.

FIG. 2 is a diagrammatic representation of a single-stage particleforming process of the invention.

FIG. 3 is a diagrammatic representation of a two-stage swelling particleforming process of the invention, including an indication of exemplarymonomers and chain transfer agent.

FIG. 4 is a diagrammatic representation of a two-stage swelling particleforming process of the invention.

FIG. 5 illustrates the use of particles of the invention in nucleotidesequencing and/or detection.

DETAILED DESCRIPTION

Throughout the description and claims of this specification, the words“comprise” and “contain” and variations of them mean “including but notlimited to”, and they are not intended to (and do not) exclude othermoieties, additives, components, integers or steps. Throughout thedescription and claims of this specification, the singular encompassesthe plural unless the context otherwise requires. In particular, wherethe indefinite article is used, the specification is to be understood ascontemplating plurality as well as singularity, unless the contextrequires otherwise.

Features, integers, characteristics, compounds, chemical moieties orgroups described in conjunction with a particular aspect, embodiment orexample of the invention are to be understood to be applicable to anyother aspect, embodiment or example described herein unless incompatibletherewith. All of the features disclosed in this specification(including any accompanying claims, abstract and drawings), and/or allof the steps of any method or process so disclosed, may be combined inany combination, except combinations where at least some of suchfeatures and/or steps are mutually exclusive. The invention is notrestricted to the details of any embodiments disclosed herein. Theinvention extends to any novel one, or any novel combination, of thefeatures disclosed in this specification (including any accompanyingclaims, abstract and drawings), or to any novel one, or any novelcombination, of the steps of any method or process so disclosed.

The reader's attention is directed to all papers and documents which arefiled concurrently with or previous to this specification in connectionwith this application and which are open to public inspection with thisspecification, and the contents of all such papers and documents areincorporated herein by reference.

All publications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including definitions, willcontrol.

The present invention provides in an embodiment novel polymer particlesand another embodiment provides a process by which the novel particlesmay be prepared. An embodiment provides novel seed particles for use inthe process for forming the polymer particles and other embodimentsprovide methods of using the polymer particles which may be obtained bythe process.

The polymer particle forming process involves a water-in-oil emulsion,i.e. a discontinuous aqueous phase suspended in a continuous oil phase.

The polymer particle forming process described herein involves,therefore, two different particles, namely a seed particle which issubjected to a swelling and polymerization process to form a polymerparticle. The terms “seed particle” and “polymer particle” are thereforeused herein as follows:

“Seed particle” means, unless the context requires otherwise, a particleobtainable by dispersion polymerization and used as an intermediate inthe polymer particle forming process.

“Polymer particle” refers to a particle which may be made from the seedparticle by suspension polymerization in the process described herein.

The mention of “hydrogel” with reference to polymer, for example polymerparticles, means a polymer gel in which the swelling agent is water. Ahydrogel polymer may absorb at least 20% of its weight in water. Ahydrogel polymer may absorb at least 45%, at least 65%, at least 85%, atleast 100% or at least 300% of its weight in in water. For example ahydrogel polymer may absorb at least 1000%, at least 1500% or even atleast 2000% of its weight in water.

The mention of “transparent” in relation to polymer particles (forexample hydrogel polymer particles) means that the particles are porousand that molecules or other reagents of interest are able to diffusereadily through aqueous solution in the pores of the particles. Forexample, crosslinked hydrogel polymer particles of the presentdisclosure may be transparent to oligonucleotides and nucleic acidamplification and sequencing reagents, e.g. the oligonucleotides may belocated partly or wholly within the pores, even when a polymerase isattached to the oligonucleotide.

The mention in this specification of “average” diameters unlessotherwise specified refers to the mode diameter for cross-linked polymerparticles (e.g. cross-linked hydrogel polymer particles) or refers tothe z-average diameter for seed particles. The mode diameter may bemeasured by disc centrifuge, for example by a CPS disc centrifuge. Thez-average diameter may be the z-average mean diameter measured bydynamic light scattering (also known as photon correlationspectroscopy). Across the entire scope, there are further herebydisclosed embodiments in which the average diameters are the modediameter, e.g. the mode diameter as determined by optical microscopy.

The term “monodisperse” means that for a plurality of particles (e. g.at least 100, more preferably at least 1000) the particles have acoefficient of variation (CV) or % polydispersity of their diameters ofless than 20%, for example less than 15%, typically of less than 10% andoptionally of less than 8%, e.g. less than 5%.

The term “Mw” is the weight average molecular weight of a polymer. It isdefined by the following formula:

${Mw} = \frac{\sum{N_{i}M_{i}^{2}}}{\sum{N_{i}M_{i}}}$

where M_(i) is the molecular weight of a particular chain and Ni is thenumber of chains of that molecular weight. The Mw may be measured usinggel permeation chromatography (GPC) relative to a set of standardpolymers with a specified eluent solvent system. For example, the Mw ofthe oligomers or polymers in seed particles may be measured by GPCrelative to polystyrene standards using as eluent DMF with 0.01 M LiBr.

The mention in this specification of “polydispersity” or “%polydispersity” refer to a value for dynamic light scattering dataderived from the “polydispersity index”. The polydispersity index is anumber calculated from a simple 2 parameter fit to the correlation data,e.g. dynamic light scattering data, as defined in ISO standard document13321:1996 E and ISO 22412:2008. The polydispersity index isdimensionless and scaled such that values smaller than 0.05 are rarelyseen other than with highly monodisperse standards. Polydispersity indexvalues of greater than 0.7 for a sample of particles indicate that thesample has a very broad size distribution, e.g. the particles are notmonodisperse.

The term “alkyl” and “C_(x)-C_(y) alkyl” (where x is at least 1 and lessthan 10, and y is a number greater than 10) as used herein includereference to a straight or branched chain alkyl moiety having, e.g. 1,2, 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms. The term includes referenceto, for example, methyl, ethyl, propyl (n-propyl or isopropyl), butyl(n-butyl, sec-butyl or tert-butyl), pentyl, hexyl and the like. Inparticular, alkyl may be a “C₁-C₆ alkyl”, i.e. an alkyl having 1, 2, 3,4, 5 or 6 carbon atoms; or a “C₁-C₄ alkyl”, i.e. an alkyl having 1, 2, 3or 4 carbon atoms. The term “lower alkyl” includes reference to alkylgroups having 1, 2, 3 or 4 carbon atoms. An alkyl may be optionallysubstituted, where chemically possible, by 1 to 5 substituents which areeach independently at each occurrence selected from: oxo, ═NR^(a),═NOR^(a), halo, nitro, cyano, NR^(a)R^(a), NR^(a)S(O)₂R^(a),NR^(a)CONR^(a)R^(a), NR^(a)CO₂R^(a), OR^(a); SR^(a), S(O)R^(a),S(O)₂OR^(a), S(O)₂R^(a), S(O)₂NR^(a)R^(a), CO₂R^(a) C(O)R^(a),CONR^(a)R^(a), C₁-C₄-alkyl, C₂-C₄-alkenyl, C₂-C₄-alkynyl, C₁-C₄haloalkyl; wherein R^(a) is independently at each occurrence selectedfrom: H and C₁-C₄ alkyl.

The term “log P” as used herein refers to the octanol-water partitioncoefficient (log P_(oct/wat)) for a compound, for example a hydrophilicvinylic monomer. The log P for a compound may be determined by any oneof a variety of methods. In particular, for compounds of use inembodiments and other compounds disclosed herein, log P may be measuredin accordance with the shake-flask method described in J. Sangster,“Octanol-water partition coefficients of simple organic compounds”, J.Phys. Chem. Ref. Data, Vol. 18, No. 3, 1989, 1111-1227 (incorporatedherein by reference in its entirety) at section 2.1.a on page 1116. LogP may also be calculated from the structure of the compound usingsoftware, e.g. log P may be calculated using ChemBioDraw® Ultra 14.0from CambridgeSoft Corp.

The Polymer Particle

The invention includes embodiments in which the particles are polymericand monodisperse. The invention includes embodiments in which theparticles are porous.

The particles may be in a population of at least 100, e.g. at least1000. For example, for the purposes of measurement the particles may bein a population of at least 100, e.g. at least 1000. For example, incertain end use applications, the particles may conveniently be in apopulation of at least 100, e.g. at least 1000.

By “monodisperse” is meant that for a plurality of particles (e. g. atleast 100, more preferably at least 1000) the particles have acoefficient of variation (CV) of their diameters of less than 20%, forexample less than 15%, typically of less than 10% and optionally of lessthan 8%, e.g. less than 5%. A particular class of polymer particles hasa CV of less than 10%. CV is defined as 100 times (standard deviation)divided by average where “average” is mean particle diameter andstandard deviation is standard deviation in particle size. The inventionalso includes embodiments where the “average” is either the z-average ormode particle diameter. CV is preferably calculated on the main mode.Thus some particles below or above mode size may be discounted in thecalculation which may for example be based on about 90% of totalparticle number (of detectable particles that is). Such a determinationof CV is performable on a CPS disc centrifuge.

The polymer particles may be produced by a polymer particle formingprocess described later in the specification, e.g. by using monodisperseseed particles as described herein.

Reverting now to the polymer particles, this specification disclosescrosslinked hydrogel polymer particles. It is a characteristic ofcrosslinked hydrogel polymer particles that, when placed in an aqueoussolution that is a good solvent for the polymer, the particles swellinstead of dissolving. By way of example, water is a good solvent forhydrogel particles comprising acrylamide polymer. Hydrogel particles arehydrophilic and swell in water and may be made in a variety of differentporosities. The crosslinked hydrogel particles disclosed herein providea combination of low nonspecific binding, monodispersity and porositiesthat provide advantages when the particles are used in biologicalassays.

The level of crosslinking in a polymer particle made by the process canbe expressed as the percentage by weight (% wt) of crosslinker monomerincluded in the total monomer used in the suspension polymerisation. The% wt of crosslinker monomer may be equivalent to the % wt of thecrosslinker in matrix polymer (i.e. the % wt of crosslinker in the dryweight of the crosslinked polymer particles). Thus, where the monomersused in the suspension polymerisation are, for example, a monofunctionalacrylamide and a bifunctional acrylamide the percentage of bifunctionalacrylamide (the crosslinker monomer) is calculated as weight percentbased upon the total weight of bifunctional acrylamide plusmonofunctional acrylamide. Typical levels of crosslinking include >1% wtcrosslinker, for example >2% wt crosslinker, e.g. >5% wt crosslinker.For example, the level of crosslinking may be >10% wt crosslinker,or >15% wt crosslinker, e.g. >20% wt crosslinker or >25% wt crosslinker.The level of crosslinking may also be, for instance 5-60% wtcrosslinker, for example 10-50% wt crosslinker, e.g. 20-40% wtcrosslinker or 20-30% wt crosslinker. The level of crosslinking may alsobe 1-30% wt crosslinker, for example 1-20% wt crosslinker, e.g. 1-10% wtcrosslinker; or the level of crosslinking may also be 2-30% wtcrosslinker, for example 2-20% wt crosslinker, e.g. 2-10% wtcrosslinker; levels which, e.g. are suitable for porous hydrogelparticles.

The level of crosslinking may be >30% wt crosslinker or >40% wtcrosslinker, for example in highly crosslinked particles. The level ofcrosslinking may be 10-90% wt crosslinker, 20-80% wt crosslinker or25-75% wt crosslinker, e.g. 25-60% wt crosslinker or 30-50% wtcrosslinker. In highly crosslinked particles the level of crosslinkingmay be up to 100% wt crosslinker, for example the hydrophilic vinylicmonomer may be a crosslinker.

As stated above, crosslinked particles swell when placed in a goodsolvent for the polymer. The amount of swelling, e.g. measured as anincrease in diameter, is related to the level of crosslinking. Particleswith a higher degree of crosslinking will typically swell less thanparticles made from a similar polymer, but with a lower degree ofcrosslinking. This property can be used to determine the relative levelof crosslinking in a sample of polymer particles by comparing the samplewith a series of standards of known, different levels of crosslinking.

The particles suitably comprise an addition polymer made by polymerisingone or more vinylic unsaturated monomers. The vinylic unsaturatedmonomers may comprise a generalised vinyl group, —CR═CH₂, where R is Hor alkyl (e.g. where R is —CH₃ or —CH₂CH₃). The vinylic unsaturatedmonomers may comprise a vinyl group, —CH═CH₂. The monomers may behydrophilic vinylic monomers, for example vinylic monomers with a log Pvalue of less than about 1, e.g. a log P value of less than about 0.5.The hydrophilic vinylic monomers may comprise a generalised vinyl group,—CR═CH₂, where R is H or alkyl (e.g. where R is —CH₃ or —CH₂CH₃). Thehydrophilic vinylic monomers may comprise a vinyl group, —CH═CH₂. Themonomers may be vinylic monomers with a log P of less than about 0.6.The monomers may be vinylic monomers with a log P of less than about0.5. For example, the monomers may be vinylic monomers with a log P ofless than about 0.3 or of less than about 0.2, e.g. the monomers may bevinylic monomers with a log P of less than about 0.1. The monomers maybe vinylic monomers with a log P of less than about 0, e.g. with a log Pof less than about −0.2. The monomers may be vinylic monomers that havea log P of from 0.5 to −2, for example a log P of from 0 to −2, e.g. alog P of from −0.2 to −2. In particular, the monomers may be vinylicmonomers that also comprise a hydrophilic group, for example anacrylamide monomer or an acrylate monomer.

The monomer used in the particles may be at least one compound offormula (I):

wherein:R¹ is —H, —CH₃ or —CH₂CH₃;R² is —OR³ or —N(R⁴)R⁵;R³ is —H, —C₁-C₆ alkyl, or —C₁-C₆ alcohol; andR⁴ and R⁵ are each independently selected from —H, —C₁-C₆ alkyl, —C₁-C₆haloalkyl, —C₁-C₆ alcohol.

Where R³ is —C₁-C₆ alkyl or —C₁-C₆ alcohol, the alkyl or alcohol may besubstituted, where chemically possible, by 1 to 5 (e.g. 1, 2, 3 or 4)substituents which are each independently at each occurrence selectedfrom: oxo, ═NR^(a), ═NOR^(a), halo, nitro, cyano, NR^(a)R^(a),NR^(a)S(O)₂R^(a), NR^(a)CONR^(a)R^(a), NR^(a)CO₂R^(a), OR^(a); SR^(a),S(O)R^(a), S(O)₂OR^(a), S(O)₂R^(a), S(O)₂NR^(a)R^(a), CO₂R^(a)C(O)R^(a), CONR^(a)R^(a), C₁-C₄-alkyl, C₂-C₄-alkenyl, C₂-C₄-alkynyl,C₁-C₄ haloalkyl; wherein R^(a) is independently at each occurrenceselected from: H and C₁-C₄ alkyl. For example, where R³ is —C₁-C₆ alkylor —C₁-C₆ alcohol, the alkyl or alcohol may be substituted, wherechemically possible, by 1 to 5 (e.g. 1, 2, 3 or 4) substituents whichare each independently at each occurrence selected from OR^(a) orCO₂R^(a), optionally where R^(a) is H.

Where R⁴ and/or R⁵ are —C₁-C₆ alkyl, —C₁-C₆ haloalkyl, —C₁-C₆ alcohol,each may be independently substituted, where chemically possible, by 1to 5 (e.g. 1, 2, 3 or 4) substituents which are each independently ateach occurrence selected from: oxo, ═NR^(a), ═NOR^(a), halo, nitro,cyano, NR^(a)R^(a), NR^(a)S(O)₂R^(a), NR^(a)CONR^(a)R^(a),NR^(a)CO₂R^(a), OR^(a); SR^(a), S(O)R^(a), S(O)₂OR^(a), S(O)₂R^(a),S(O)₂NR^(a)R^(a), CO₂R^(a) C(O)R^(a), CONR^(a)R^(a), C₁-C₄-alkyl,C₂-C₄-alkenyl, C₂-C₄-alkynyl, C₁-C₄ haloalkyl; wherein R^(a) isindependently at each occurrence selected from: H and C₁-C₄ alkyl. Forexample, where R⁴ and/or R⁵ are —C₁-C₆ alkyl, —C₁-C₆ haloalkyl, —C₁-C₆alcohol, each may be independently substituted, where chemicallypossible, by 1 to 5 (e.g. 1, 2, 3 or 4) substituents which are eachindependently at each occurrence selected from OR^(a) or CO₂R^(a),optionally where R^(a) is H.

R¹ may be —H or —CH₃. For example, R¹ may be —H.

R² may be —OR³. R² may be —N(R⁴)R⁵.

R³ may be —H. R³ may be —C₁-C₆ alkyl. For example, R³ may be —C₁-C₆alkyl substituted by 1 or 2 substituents which are each independently ateach occurrence selected from: oxo, halo, cyano, NR^(a)R^(a),NR^(a)CONR^(a)R^(a), NR^(a)CO₂R^(a), OR^(a); CO₂R^(a) C(O)R^(a),CONR^(a)R^(a), C₁-C₄-alkyl, C₂-C₄-alkenyl, C₂-C₄-alkynyl, C₁-C₄haloalkyl; wherein R^(a) is independently at each occurrence selectedfrom: H and C₁-C₄ alkyl, for example wherein R^(a) is —H. R³ may be—C₁-C₆ alcohol. For example, R³ may be —C₁-C₆ alcohol substituted by 1or 2 substituents which are each independently at each occurrenceselected from: oxo, halo, cyano, NR^(a)R^(a), NR^(a)CONR^(a)R^(a),NR^(a)CO₂R^(a), OR^(a); CO₂R^(a) C(O)R^(a), CONR^(a)R^(a), C₁-C₄-alkyl,C₂-C₄-alkenyl, C₂-C₄-alkynyl, C₁-C₄ haloalkyl; wherein R^(a) isindependently at each occurrence selected from: H and C₁-C₄ alkyl, forexample wherein R^(a) is —H.

R⁴ may be —H or —C₁-C₆ alkyl. R⁴ may be —C₁-C₆ alkyl. For example, R⁴may be —C₁-C₆ alkyl substituted by 1 or 2 substituents which are eachindependently at each occurrence selected from: oxo, halo, cyano,NR^(a)R^(a), NR^(a)CONR^(a)R^(a), NR^(a)CO₂R^(a), OR^(a); CO₂R^(a)C(O)R^(a), CONR^(a)R^(a), C₁-C₄-alkyl, C₂-C₄-alkenyl, C₂-C₄-alkynyl,C₁-C₄ haloalkyl; wherein R^(a) is independently at each occurrenceselected from: H and C₁-C₄ alkyl, for example wherein R^(a) is —H.

R⁵ may be —H or —C₁-C₆ alkyl. R⁵ may be —C₁-C₆ alkyl. For example, R⁵may be —C₁-C₆ alkyl substituted by 1 or 2 substituents which are eachindependently at each occurrence selected from: oxo, halo, cyano,NR^(a)R^(a), NR^(a)CONR^(a)R^(a), NR^(a)CO₂R^(a), OR^(a); CO₂R^(a)C(O)R^(a), CONR^(a)R^(a), C₁-C₄-alkyl, C₂-C₄-alkenyl, C₂-C₄-alkynyl,C₁-C₄ haloalkyl; wherein R^(a) is independently at each occurrenceselected from: H and C₁-C₄ alkyl, for example wherein R^(a) is —H.

The compound of formula (I) may have a log P value of less than about 1,e.g. a log P of less than about 0.5. The compound of formula (I) mayhave a log P of less than about 0.6. The compound of formula (I) mayhave a log P of less than about 0.5. For example, the compound offormula (I) may have a log P of less than about 0.3 or of less thanabout 0.2, e.g. the compound of formula (I) may have a log P of lessthan about 0.1. The compound of formula (I) may have a log P of lessthan about 0, e.g. with a log P of less than about −0.2. The compound offormula (I) may have a log P of from 0.5 to −2, for example a log P offrom 0 to −2, e.g. a log P of from −0.2 to −2.

The monomer may comprise at least one hydrophilic vinylic monomercomprising a primary amide group (—C(O)NH₂).

Acrylamide monomers and/or acrylate monomers may be mentioned inparticular. Suitable monomers include acrylamide (prop-2-enamide),N-(hydroxymethyl)acrylamide, N-hydroxyethyl acrylamide,N-[tris(hydroxymethyl)methyl]acrylamide, 3-acrylamidopropanoic acid,methacrylamide, N-(2-hydroxyethyl)methacrylamide,N-(3-aminopropyl)methacrylamide, hydroxypropylacrylamide,N,N-dimethylacrylamide, 2-hydroxyethyl acrylate, 2-hydroxyethylmethacrylate, acrylic acid; and other acrylamide monomers, acrylicmonomers, methacrylamide monomers, or methacrylic monomers with a log Pvalue of less than about 1 (e.g. with a log P value of less than about0.5).

The monomer may comprise a mixture of monomers. For example, the monomermay comprise at least one monomer as defined above and at least onecompatible functional monomer. A corresponding functional monomer is ahydrophilic vinylic monomer as defined herein, which comprises acarboxylic acid (—COOH), a primary amine or a secondary amine.

In highly crosslinked particles, the monomer may be or comprise acrosslinker, e.g. a crosslinker as defined elsewhere herein. Forexample, the monomer may be or comprise at least one compound of formula(IIa) or (IIb).

A functional monomer may be a vinylic monomer with a log P value of lessthan about 1, e.g. a log P of less than about 0.5, which comprises acarboxylic acid or primary amine. A functional monomer may be a vinylicmonomer with a log P of less than about 0.6. A functional monomer may bea vinylic monomer with a log P of less than about 0.5. For example, thefunctional monomer may be a vinylic monomer with a log P of less thanabout 0.3 or of less than about 0.2, e.g. the functional monomer may bea vinylic monomer with a log P of less than about 0.1. A functionalmonomer may be a vinylic monomer with a log P of less than about 0, e.g.with a log P of less than about −0.2. A functional monomer may be avinylic monomer that has a log P of from 0.5 to −2, for example a log Pof from 0 to −2, e.g. a log P of from −0.2 to −2. A functional monomermay be a compound of formula (I) which comprises a carboxylic acid orprimary amine. A functional monomer may be an acrylamide monomer whichcomprises a carboxylic acid or primary amine. Suitable functionalmonomers include 3-acrylamidopropionic acid, 4-acrylamidobutanoic acid,5-acrylamidopentanoic acid, N-(3-aminopropyl)methacrylamide, and acrylicacid.

Where at least one functional monomer is present, the amount of thefunctional monomer may be about 0.1 to about 100% mol, for example about0.2 to about 50% mol, e.g. about 0.5 to about 40% mol or about 1 toabout 30% mol (such as about 2 to about 20 mol %. The amount offunctional monomer may be about 0.1 to about 10% wt, for example about0.2 to about 5% wt, e.g. about 0.5 to about 2% wt. The % wt may refer tothe percent by weight of the functional monomer included in the totalmonomer used in the polymerisation process. The total monomer may, forexample, comprise a hydrophilic vinylic monomer that is not the at leastone functional monomer, a crosslinker and a functional monomer.

Crosslinking may be achieved by incorporating a crosslinker comprisingat least two (e.g. two) vinyl groups (—CH═CH₂) as a comonomer. Thecrosslinker may be at least one compound of formula (IIa) or (IIb):

wherein R⁶ is selected from —C₁-C₆ alkyl-, —C₁-C₆ heteroalkyl-, —C₁-C₆cycloalkyl-, —C₁-C₆ hydroxyalkyl-, —C₁-C₀ ether-, polyether comprising 2to 100 C₂-C₃ ether units;R⁷ and R⁸ are each independently selected from —H, —C₁-C₆ alkyl, —C₁-C₆heteroalkyl, —C₃-C₆ cycloalkyl, —C₁-C₆ hydroxyalkyl, or —C₁-C₆ ether;R⁹ is —N(R¹¹)C(O)CH═CH₂;R¹⁰ is selected from —H and —N(R¹²)C(O)CH═CH₂; andR¹¹ and R¹² are each independently selected from —H, —C₁-C₆ alkyl,—C₁-C₆ heteroalkyl, —C₃—C cycloalkyl, —C₁-C₆ hydroxyalkyl, or —C₁-C₆ether.

The crosslinker may be at least one compound of formula (IIa). Thecrosslinker may be at least one compound of formula (IIb).

R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹ and R¹² may be independently substituted, wherechemically possible, by 1 to 5 substituents which are each independentlyat each occurrence selected from: oxo, ═NR^(a), ═NOR^(a), halo, nitro,cyano, NR^(a)R^(a), NR^(a)S(O)₂R^(a), NR^(a)CONR^(a)R^(a),NR^(a)CO₂R^(a), OR^(a); SR^(a), S(O)R^(a), S(O)₂OR^(a), S(O)₂R^(a),S(O)₂NR^(a)R^(a), CO₂R^(a) C(O)R^(a), CONR^(a)R^(a), C₁-C₄-alkyl,C₂-C₄-alkenyl, C₂-C₄-alkynyl, C₁-C₄ haloalkyl; wherein R^(a) isindependently at each occurrence selected from: H, C₁-C₄ alkyl and C₁-C₄alkenyl.

R⁶ is selected from —C₁-C₆ alkyl-, —C₁-C₆ heteroalkyl-, —C₁-C₆cycloalkyl-, —C₁-C₆ hydroxyalkyl- and —C₁-C₆ ether-. R⁶ may be selectedfrom —C₁-C₆ alkyl- and —C₁-C₆ hydroxyalkyl-. R⁶ may be a —C₁-C₆ alkyl-,for example —CH₂—, —(CH₂)₂—, —(CH₂)₃—, or —(CH₂)₄—, e.g. —(CH₂)₂—. R⁶may be a —C₁-C₆ hydroxyalkyl-, for example —C(OH)H—, —(C(OH)H)₂—,—(C(OH)H)₃—, or —(C(OH)H)₄—, e.g. —(C(OH)H)₂—.

R⁶ may be a —C₁-C₆ heteroalkyl-, optionally wherein the heteroatom is anamine (e.g. a tertiary amine). For example a —C₁-C₆heteroalkyl-substituted by C(O)R^(a) on the hetero atom, optionallywherein the heteroatom is an amine, e.g. R⁶ may be—CH₂CH₂N(C(O)CH═CH₂)CH₂CH₂—.

Where R⁶ is a polyether, the polyether may be linear or branched. R⁶ maybe a polyether comprising 2 to 100 C₂-C₃ ether units, e.g. a polyethercomprising 2 to 50 C₂-C₃ ether units. R⁶ may be a polyether comprising 2to 100 C₂ ether units, e.g. a polyether comprising 2 to 50 C₂ etherunits. For example R⁶ may be —(CH₂)_(r)(OCH₂CH₂)_(n)O(CH₂)_(s), whereinr and s are each independently 2 or 3 (e.g. 2); and n is an integer from1 to 100 (e.g. 1 to 50 or 1 to 25; such as 5 to 50 or 5 to 25). Withoutwishing to be bound by any theory, it is believed that crosslinkerscomprising a polyether (e.g. where R⁶ is a polyether) have excellentsolubility in the aqueous phase. This means that, while suchcrosslinkers may be used to provide particles with a low level ofcrosslinking (e.g. 1-20% wt crosslinker, or 1-10% wt crosslinker), suchpolyether comprising crosslinkers are particularly suitable forproviding particles comprising relatively high levels of crosslinking,for example >20% wt crosslinker, >25% wt crosslinker, or >30% wtcrosslinker. For example, the level of crosslinking may be 10-90% wtcrosslinker, 20-80% wt crosslinker or 25-75% wt crosslinker, e.g. 25-60%wt crosslinker or 30-50% wt crosslinker.

R⁷ and/or R⁸ and/or R¹¹ and/or R¹² may be H. For example, R⁷ and/or R⁸may be H. For example, R¹¹ and/or R¹² may be H.

The compound of formula (II) may have a log P value of less than about1, e.g. a log P of less than about 0.5. The compound of formula (II) mayhave a log P of less than about 0.6. The compound of formula (II) mayhave a log P of less than about 0.5. For example, the compound offormula (II) may have a log P of less than about 0.3 or of less thanabout 0.2, e.g. the compound of formula (II) may have a log P of lessthan about 0.1. The compound of formula (II) may have a log P of lessthan about 0, e.g. with a log P of less than about −0.2. The compound offormula (II) may have a log P of from 0.5 to −2, for example a log P offrom 0 to −2, e.g. a log P of from −0.2 to −2.

Exemplary crosslinkers of use in particles of the invention includeN,N′-(1,2-dihydroxyethylene)bisacrylamide,N,N′-methylenebis(acrylamide), N,N′-ethylenebis(acrylamide), piperazinediacrylamide, glycerol 1,3-diglycerolate diacrylate,N,N′-((ethane-1,2-diylbis(oxy))bis(ethane-2,1-diyl))diacrylamide,polyethyleneglycol diacrylamide (MW≤2000), 4-arm PEG-acrylamide(MW≤2000), N,N-bis(2-acrylamidoethyl)acrylamide. The exemplarycrosslinkersN,N′-((ethane-1,2-diylbis(oxy))bis(ethane-2,1-diyl))diacrylamide,polyethyleneglycol diacrylamide (MW≤2000) and 4-arm PEG-acrylamide(MW≤2000), (in particularN′-((ethane-1,2-diylbis(oxy))bis(ethane-2,1-diyl))diacrylamide) areparticular suitable for use in highly crosslinked particles andmechanically more stable particles, for example particles with a levelof crosslinking of at least 20% wt crosslinker (e.g. a level ofcrosslinking of at least 30% wt crosslinker). An embodiment of theinvention includes the use of a combination of crosslinkers. As aparticular monomer may be mentioned acrylamide (prop-2-en-amide), forwhich N,N′-(1,2-dihydroxy bisacrylamide) is a suitable crosslinker. As aparticular monomer may be mentioned hydroxymethyl acrylamide, for whichN,N′-((ethane-1,2-diylbis(oxy))bis(ethane-2,1-diyl))diacrylamide is asuitable crosslinker. As a particular monomer may be mentionedhydroxyethyl acrylamide, for whichN,N′-((ethane-1,2-diylbis(oxy))bis(ethane-2,1-diyl))diacrylamide is asuitable crosslinker.

The crosslinker may be a compound that does not comprise a primaryamine, secondary amine, hydroxy or carboxylic acid. The crosslinker maybe a compound of formula (IIa) or formula (IIb) that does not comprise aprimary amine, secondary amine, hydroxyl or carboxylic acid.

A number of exemplary monomers and crosslinkers that may be used are setout in Table 1. A number of other monomers are set out in Table 2. Thelog P values for the monomers listed in table 1 and table 2 weredetermined using the software ChemBioDraw® Ultra 14.0 from CambridgeSoftCorp. In some particles that are embodiments of the invention themonomers of Table 2 are not used, for example due to the log P values ofthe monomers. In addition, the monomers methyl acrylate and methacrylicacid of Table 2 may be used in conventional Ugelstad procedures,therefore these monomers and other monomers with higher log P values maybe less preferred for use in the particles of the present invention,which may be made with a polymer particle forming process describedherein.

TABLE 1 Exemplary Monomers and crosslinkers Name Abbreviation Structurelog P Function Acrylamide AAm

−0.27 Monomer N-(Hydroxymethyl)acrylamide HMAAm

−0.28 Monomer N-Hydroxyethyl acrylamide HEAAm

−0.56 Monomer N-[Tris(hydroxymethyl)methyl] acrylamide THMAAm

−1.73 Monomer 3-acrylamidopropanoic acid AAmPA

−0.48 Functional monomer Methacrylamide MAAm

 0.08 Monomer N-(2-hydroxyethyl)methacryl- amide HEMAAm

−0.21 Monomer N-(3-Aminopropyl)methacryl- amide APMAAm

−0.59 Functional monomer 2-Hydroxyethyl acrylate HEA

 0.12 Monomer 2-Hydroxyethyl methacrylate HEMA

 0.47 Monomer Acrylic acid AA

 0.38 Functional monomer N,N′-(1,2-Dihydroxyethylene) bisacrylamidDHEBAAm

−0.47 Crosslinker N,N′-Methylenebis(acrylamide) MBAAm

−0.04 Crosslinker N,N′-Ethylenebis(acrylamide) EBAAm

−0.32 Crosslinker Piperazine diacrylamide PDAAm

−0.09 Crosslinker Glycerol 1,3-diglycerolate diacrylate GDGDA

−0.89 Crosslinker N,N′-((ethane-1,2-diylbis(oxy))bis(ethane-2,1-diyl))diacrylamide EGBEAAm

−0.63 Cross-linker Polyethyleneglycol diarcylamide (MW ≤2000) PEGDAAm

≤−0.63* Cross-linker 4-Arm PEG-Acrylamide (MW ≤2000) 4PEGAAm

≤−0.63* Cross-linker N,N-bis(2-acrylamidoethyl) acrylamide BAAmEAAm

−0.37 Cross-linker hydroxypropylacrylamide HPAAm

−0.45 Monomer N,N-dimethylacrylamide DMAAm

 0.2 Monomer

TABLE 2 Other Monomers and crosslinkers Name Abbreviation Structure logP Function N-isopropylacrylamide NiPAm

0.62 Monomer Methyl acrylate MA

0.64 Monomer Methacrylic acid MAA

0.73 Functional monomer Propargyl acrylate PA

0.86 Monomer Methyl methacrylate MMA

0.99 Monomer Chloromethyl acrylate CMA

1.16 Monomer Vinyl acrylate VA

1.22 Monomer N,N′-hexamethylene-bis- acrylamide HMBAAm

1.07 Cross-linker Ethyleneglycol dimetharcylate EGDMA

1.74 Cross-linker

The level of crosslinking in the crosslinked hydrogel polymer particlescan be expressed as the percentage by weight (% wt) of crosslinkermonomer included in the total monomer used in the polymerisation.Typical levels of crosslinking include >1% wt crosslinker, forexample >2% wt crosslinker, e.g. >5% wt crosslinker. For example, thelevel of crosslinking may be >10% wt crosslinker, or >15% wtcrosslinker, e.g. >20% wt crosslinker (such as >30% wt crosslinkeror >40 wt % crosslinker). The level of crosslinking may also be 1-60% wtcrosslinker, e.g. 5-30% wt crosslinker. For example, the level ofcrosslinking may be 5-60% wt crosslinker, for example 10-50% wtcrosslinker. The level of crosslinking may also be, for instance 15-40%wt crosslinker, for example 20-40% wt crosslinker, e.g. 20-30% wtcrosslinker. The level of crosslinking may be 10-90% wt crosslinker,20-80% wt crosslinker or 25-75% wt crosslinker, e.g. 25-60% wtcrosslinker or 30-50% wt crosslinker; for example in highly crosslinkedparticles. The level of crosslinking may also be 1-30% wt crosslinker,for example 1-20% wt crosslinker, e.g. 1-10% wt crosslinker. The levelof crosslinking may also be 2-30% wt crosslinker, for example 2-20% wtcrosslinker, e.g. 2-10% wt crosslinker. In highly crosslinked particlesthe level of crosslinking may be up to 100% wt crosslinker, for examplethe hydrophilic vinylic monomer may be a crosslinker.

In an embodiment, the particles may have an average diameter of at least500 nm, e.g. at least 600 nm, optionally at least 800 nm, as in the caseof particles having a diameter of at least 1 μm.

In an embodiment, the particles may have an average diameter of not morethan 10 μm, e.g. not more than 5 μm, optionally not more than 3 μm, asin the case of particles having a diameter of not more than 2 μm.

In an embodiment, the invention includes a class of polymer particleshaving average diameters of from 0.5 μm to 10 μm, e.g. of from 0.8 μm to5 μm.

The size and size distribution of the polymer particles may bedetermined as described below under the heading “analytical methods”.

The disclosure includes porous polymer particles having a porosity of atleast 5% e.g. at least 10%. The disclosure includes porous particleshaving a porosity of from 20% to 95%, particularly of from 30% to 90%,e.g. of from 40% to 90%, such as from 50% to 80%.

Porosity may be calculated after determining the density of the hydrogelpolymer particles, as acrylamide and acrylate polymer has a density ofabout 1.3 g/mL, while water has a density of about 1 g/mL. The porouspolymer particles may be transparent to solvated molecules, for examplethe porous polymer particles may be transparent to oligonucleotides andnucleic acid amplification reagents and sequencing reagents (e.g.primers, nucleotides and polymerases).

In an embodiment, the particles comprise functional groups. Thefunctional groups may be selected from a hydroxyl group, carboxylic acid(—COOH), a primary amine or a secondary amine. In an embodiment, thefunctional groups are provided by a hydrophilic vinylic monomer (e.g.compound of formula (I)) and not a crosslinker (e.g. compound of formula(IIa) or formula (IIb)). The functional groups may be enhanced tofacilitate binding with target analytes (e.g. oligonucleotides) ortarget receptors. Exemplary methods of enhancing functional groups ofparticles of the invention are described herein in the section relatingto “Uses of the Particles”.

In an embodiment, the particles comprise one or more oligonucleotidesattached to the particles. The oligonucleotides may be attached to theparticles via a linker. For example, each of (or a proportion of) theparticles may comprise a plurality of oligonucleotides attached to theparticle. The plurality of oligonucleotides may be identical for eachindividual particle. For example, a first particle may comprise aplurality of oligonucleotides having a first sequence attached to thefirst particle, and a second particle may comprise a plurality ofoligonucleotides having a second sequence attached to the secondparticle. In an embodiment where the particles of the invention areporous, the oligonucleotides may be attached to the outer surface of theparticle, or attached inside a pore, or attached throughout the particlematrix. The pores may be of sufficient size to render the particletransparent to the oligonucleotides, related molecules and otherreagents, such that the oliogonucleotides may be located partly orwholly within the pores, even when a polymerase is attached to theoligonucleotide.

The particles of the invention may be stable (i.e. resistant to polymerdegradation) in aqueous solution over the typical temperature ranges ofaqueous solutions. The monodisperse crosslinked hydrogel polymerparticles may be stable at a temperature of up to about 100° C. Forexample the monodisperse crosslinked hydrogel polymer particles may bestable in a temperature range of from about 0° C. to about 100° C.

Seed Particles

The polymer particles may be prepared by a polymer particle formingprocess, starting with specific seed particles. The applicant hasidentified that the seed particles typically used in other processes,e.g. polystyrene seed particles, are not compatible with the process ofthe invention. The invention therefore provides seed particles that aresuitable for use in a polymer particle forming process of the invention.

In an embodiment the seed particles are monodisperse. The seed particlescomprise a plurality of non-crosslinked oligomers of polyN,N-dimethylacrylamide and a z-average diameter of from 100 nm to 1,500nm. Each of the seed particles may comprise more than 1×10⁵ (e.g. morethan 1×10⁶) non-crosslinked oligomers of poly N,N-dimethylacrylamide,for example each of the seed particles may comprise more than 5×10⁶non-crosslinked oligomers of poly N,N-dimethylacrylamide.

The seed particles may have a z-average diameter of from 150 nm to 1,300nm. For example, the seed particles may have a z-average diameter of 300nm to 1,100 nm.

The oligomers have a weight average molecular weight (Mw) of from about2,000 Da to about 100,000 Da when measured by GPC relative topolystyrene standards. Additionally or alternatively to the specified Mwrange, the polymer of each oligomer may comprise about 30 to 2,000 (e.g.about 60 to about 1,000, or about 80 to about 500) monomer units.

The Mw of the seed particle oligomers may be less than 100,000 Da,optionally less than 50,000 Da, further optionally less than 40,000 Da,e.g. less than 30,000 Da. The Mw may be more than 4,000 Da, optionallymore than 5,000 Da, further optionally more than 6,000 Da, as in thecase of more than 8,000 Da, e.g. more than 10,000 Da. For example, theMw may be from 5,000 Da to 70,000 Da, e.g. from 6,000 Da to 60,000 Da,for example from 7,000 Da to 50,000 Da or from 8,000 Da to 40,000 Da.

Without wishing to be bound by any theory, it is believed that the Mw ofthe oligomers of the seed particles is an important characteristic ofthe seed particles. For example, it may be that having the Mw of theseed particle oligomers in the specified range is necessary to provideseed particles that form monodisperse polymer particles during theparticle forming process described herein. If the seed particle polymerhas a higher molecular weight, for example because no chain transferagent was used during seed particle formation (such as for themonodisperse microspheres of M. Babic and D. Horak, MacromolecularReaction Engineering, 2007, 1, 86-94), the particles are not suitablefor use in the present process, for example because the particlesresulting from such a process may not be monodisperse, e.g. the CV maybe greater than 20%.

The seed particles may have the characteristics of seed particles formedin accordance with the methods of forming seed particles disclosedherein. For example, the seed particles may have the characteristics ofseed particles formed by radical initiated polymerisation ofN,N-dimethylacrylamide in an organic solvent in the presence of astabilizer and a chain transfer agent.

Preparation of Seed Particles

The polymer particles may be prepared by a process as described herein,starting with specific seed particles. The invention therefore providesmethods of preparing monodisperse seed particles.

The monodisperse seed particles may be made by a method comprisingdissolving N,N-dimethylacrylamide, a stabilizer, a radical initiator anda chain transfer agent in an organic solvent to form a reaction mixture;and heating the reaction mixture to activate the initiator, therebyforming the monodisperse seed particles.

In this method, once the reaction mixture is formed in a suitablecontainer, the reaction mixture is typically mixed, for example with astirrer during the heating step.

Preferably, the polymerization reaction will occur in a reaction mixturethat comprises a minimal amount of oxygen. Thus the reaction mixture maybe purged of oxygen prior to heating the reaction mixture. The purgingmay comprise sparging with a chemically inert gas. The inert gas may benitrogen or a noble gas (e.g. helium, neon, argon, krypton or xenon).For example, the inert gas may be argon, helium or nitrogen, e.g. argonor nitrogen.

The method may be considered an inverse precipitation polymerization,with the hydrophilic seed particles precipitating out of the organicsolvent. The stabilizer may prevent aggregation of seed particles,assisting in the formation the monodisperse seed particles. Theselection of a suitable stabilizer is dependent on the ability of thestabilizer to be dissolved in the organic solvent. The stabilizer may bea block copolymer of styrene and a polyolefin, for example a triblockcopolymer based on styrene and ethylene/butylene, e.g. a linear triblockcopolymer consisting of styrene and ethylene/butylene. The stabilizermay be a triblock copolymer consisting of a midblock ofpoly(ethylene-co-butylene) and outer blocks of polystyrene. Exemplarystabilizers comprise Kraton A1535H, Kraton G1650M, Kraton G1652M, orKraton G1657M, or combinations thereof.

The radical initiator may be a peroxide initiator or an azo-initiator,for example a peroxide initiator or an azo-initiator that decomposes atelevated temperature. Exemplary radical initiators include2,2′-Azobis(2-methylpropionitrile), 2,2′-Azodi(2-methylbutyronitrile),2,2′-Azobis(2,4-dimethyl valeronitrile), dibenzoylperoxide and the like.The radical initiator may be 2,2′-Azobis(2-methylpropionitrile). Theradical initiator may be 2,2′-azodi(2-methylbutyronitrile). Thetemperature to which the reaction mixture may be heated will depend onthe temperature at which the radical initiator is activated. Heating thereaction mixture to activate the initiator may comprise heating thereaction mixture to a temperature of at least 40° C., for exampleheating to a temperature of at least 50° C., e.g. heating to atemperature of at least 60° C., or e.g. heating to a temperature of atleast 70° C. For example, when the radical initiator is2,2′-Azobis(2-methylpropionitrile), the reaction mixture maybe heated toa temperature of at least 50° C. or a temperature of at least 60° C.

The addition of a chain transfer agent reduces the molecular weight ofthe polymer of the seed particles by reacting with the free radical of agrowing polymer chain to terminate the chain and transfer the loneelectron to a radical species derived from the chain transfer agent. Theradical species derived from the chain transfer agent may then reactwith a monomer to form a radical from the monomer, which can then reactwith another monomer to commence formation of a new polymer chain. Thechain transfer reagent may be a thiol or a haloalkane. For example, thechain transfer agent may be selected from thiols (e.g. 1-octanethiol,hexane thiol, 6-mercapto-1-hexanol, benzylthiol), alkyl thiols (e.g.1-octanethiol, hexane thiol), carbon tetrachloride andbromotrichloromethane. For example, the chain transfer agent may be1-octanethiol. The total amount of chain transfer agent added can be inthe range 1 mol per 10 mol of monomer to 1 mol per 300 mol of monomer,for example 1 mol per 20 mol of monomer to 1 mol per 100 mol monomer,e.g. approximately 1 mol chain transfer agent per 30 mol of monomer. Thetime of addition of the chain transfer agent is important to obtainmonodisperse seed particles: the chain transfer agent should be presentin the reaction mixture prior to the initiation of polymerization (i.e.prior to activation of the radical initiator). This finding issurprising, as for seed particles of use in conventional Ugelstadprocesses (e.g. polystyrene seed particles), the chain transfer agent,if added, should be added after the commencement of particle formationas taught in WO 2010/125170, the content of which is incorporated hereinby reference.

The organic solvent may comprise a mixture of an alkane component and anaromatic component. The organic solvent may comprise a single component,for example an alkane component or an aromatic component. The alkanecomponent may be or comprise hexane, heptane or octane. The aromaticcomponent may be or comprise a C₁-C₁₀ alkyl substituted phenyl or a—C₁-C₆ di-alkyl substituted phenyl, for example a C₁-C₄ alkylsubstituted phenyl, e.g. toluene. Where the organic solvent comprises asingle component and that component is an aromatic component, thearomatic component may be a C₄-C₁₂ alkyl substituted phenyl, or a C₂-C₈di alkyl substituted phenyl. The alkane component may be heptane and thearomatic component may be toluene. The alkane component and aromaticcomponent may be present in a weight ratio of alkane component:aromaticcomponent of between about 0.5:1 to about 20:1, for example the weightratio of alkane component:aromatic component may be between about 1:1 toabout 15:1, e.g. between about 1:1 to about 10:1.

The reaction mixture may comprise: the N,N-dimethylacrylamide in anamount of about 2% wt to about 5% wt; the stabiliser in an amount ofabout 1% wt to about 5% wt; the radical initiator in an amount of about0.01% wt to about 4% wt (e.g. about 0.05% wt to about 0.25% wt); and thechain transfer reagent in an amount of about 0.05% wt to about 0.25% wt.

The monodisperse seed particles may then be subjected to an particleforming process, for example as outlined below:

Preparation of Particles

The present invention provides a method of forming monodispersecrosslinked hydrogel polymer particles. An Ugelstad process cannot beused to directly form such particles, for example because the Ugelstadprocess requires that the particles form in the oil phase of anoil-in-water emulsion, while hydrogel polymer particles andcorresponding hydrophilic monomers would be preferentially soluble inwater. The applicant has addressed this by providing the methods of thepresent disclosure, which provide a particle forming process that usesan aqueous (discontinuous phase) in oil (continuous phase) system. Thisis believed to represent the first such process that has been used toform monodisperse crosslinked polymer particles.

In an embodiment the present invention provides a method of formingmonodisperse crosslinked hydrogel polymer particles. The methodcomprises forming a solution (a) of at least 2% wt of a hydrophilicvinylic monomer in an aqueous solution, the aqueous solution alsocomprising a crosslinker comprising at least two vinyl groups; forming asolution (b) of stabilizer in an organic solvent, wherein the organicsolvent is not miscible in water, and wherein at least one of solution(a) and (b) comprises a radical initiator; mixing solutions (a) and (b)to form a water-in-oil emulsion (c) and adding monodisperse seedparticles to the emulsion; allowing the monodisperse seed particles toform swollen particles in the emulsion; and polymerising the swollenparticles to form the monodisperse crosslinked hydrogel polymerparticles. As explained below, this method may be considered a singlestage method, as it comprises a single step of swelling and a singlestep of polymerisation.

The solution (a) may be formed by first forming a solution of at least2% wt of the hydrophilic vinylic monomer in an aqueous solution, andthen adding the crosslinker; the crosslinker may be added before thehydrophilic vinylic monomer; or the hydrophilic vinylic monomer andcrosslinker may be added to the solution at about the same time.Similarly, the stabilizer and radical initiator may be added to solution(b) at about the same time or sequentially.

In the context of the present disclosure, an organic solvent isconsidered not miscible with water when the organic solvent and waterwould separate into two separate phases, when an amount of at least 5%wt water is mixed with the organic solvent.

This method involves swelling the seed particles with an aqueousdiscontinuous phase in an oil continuous phase. A schematic of thisprocess, which may be considered a single stage process as it comprisesa single step of swelling and single step of polymerisation, is providedin FIG. 1 (illustrated with specific monomers) and FIG. 2 (illustratedmore generally). The method illustrated in FIGS. 1 and 2 is convenientlydivided into 2 steps, swelling of the monodisperse seed particles 100 toform swollen particles 110 and polymerisation of the monomer in theswollen seed particles to form crosslinked hydrogel polymer particles120. Prior to the first step a water-in-oil emulsion is formed. Thewater-in-oil emulsion is formed by mixing an aqueous solution comprisingexemplary monomer acrylamide 101 and exemplary crosslinker 1,2-dihydroxybisacrylamide 102 with an oil phase comprising a steric stabiliser, withthe emulsion formed when the water and oil phases are agitated (e.g. bystirring). If a (very) highly crosslinked particle is desired, it isalso possible to replace the monomer with a crosslinker, so that theonly monomers present in the water-in-oil emulsion are crosslinkingmonomers (i.e. crosslinkers, e.g. 1,2-dihydroxy bisacrylamide 102). Thewater-in-oil emulsion also typically contains an initiator, which mayhave been added to the oil phase. The initiator is a compound that uponactivation will initiate polymerisation of the monomer and crosslinker.The monodisperse seed particles 100 comprise non-crosslinked oligomersof poly N,N-dimethylacrylamide 131 and are considered “activated seedparticles”. The seed particles 100 may be prepared as describedelsewhere in this application. In the first step monodisperse seedparticles 100 are added to the water-in-oil emulsion and the emulsion isagitated for a period of time (for instance for at least 30 minutes orfor at least 1 hour, typically for 4-48 hours). During this period oftime the monomer 101 and crosslinker 102 diffuse into the activated seedparticles 100 to form the swollen seed particles 110. The swollen seedparticles 110 comprise a mixture of at least the monomer 101, thecrosslinker 102 and polymer from the activated seed particles 100. Theswollen seed particles 110 may also include other components, forinstance one or more porogens, which can enter the particles if includedin the water-in-oil emulsion. For example, in the illustrated method,water is also present in the swollen seed particles 110, and this watermay be considered a porogen. The second step comprises polymerisation ofthe monomer 101 and crosslinker 102 to form the crosslinked hydrogelpolymer particles 120. In the second step polymerisation is initiated byactivating the initiator, for example by heating the emulsion.

The provision of activated seed particles is a key feature of asuccessful Ugelstad process. Activation of the seed particle istypically provided by adding an organic compound of very low solubilityas an emulsion to produce entropically activated seed particles. Theapplicant has, however, surprisingly determined that this additionalactivation step is not necessary in the process, for example where theseed particles comprise oligomers of poly N,N-dimethylacrylamide.

Swelling is key for embodiments of the particle forming process. In thisprocess, the monomers and (when present) porogen are required to have alimited solubility in the continuous phase. If the monomers and theporogen have a solubility that is too high in the continuous phase, thesolubility will not assist in driving the monomers (and optionalporogen) to enter the seeds and form swollen particles. If, on the otherhand, the monomers (and optional porogen) have insufficient solubilityin the continuous phase, there will be negligible diffusion through thecontinuous phase and thus no mass transport of monomer to the seeds arepossible, preventing the formation of swollen particles. The time tocomplete swelling, i.e. the time until all monomer (and optionalporogen) are co-localized to the seed particles, will depend highly onfactors such as temperature, solubility, and viscosity and will thusvary from system to system. Typical time scales for the step of allowingthe monodisperse seed particles to form swollen particles will thereforevary from 30 min to 48 h. For example, in an embodiment of the method,the step of allowing the monodisperse seed particles to swell may beperformed for at least 30 minutes, e.g. for at least 1 hour. The step ofallowing the monodisperse seed particles to swell may be performed forat least 4 hours, at least 8 hours or at least 12 hours. The upper limitfor the swelling time is not believe to be critical, for example it isbelieved that a swelling time of several days, e.g. 3 days or 2 days maybe used.

The step of allowing the monodisperse seed particles to form swollenparticles may comprise mixing the emulsion, e.g. mixing the emulsion forsubstantially all of the swelling time. The step of allowing themonodisperse seed particles to form swollen particles may be performedat a temperature of between about 10° C. to about 60° C., for example ata temperature of between about 10° C. to about 40° C., e.g. at atemperature of between about 15° C. to about 30° C. For example themixing may be performed at a temperature of between about 10° C. toabout 60° C. for example at a temperature of between about 10° C. toabout 40° C., e.g. at a temperature of between about 15° C. to about 30°C.

Solution (a) comprises at least 2% wt of hydrophilic vinylic monomer inan aqueous solution. The aqueous solution may be water. The aqueoussolution may comprise water and up to 50% wt (for example up to 30% wtor up to 25% wt, e.g. up to 20% wt, optionally up to 10% wt) of a watermiscible organic solvent. The water miscible organic solvent may be aC₁-C₄ alcohol, for example ethanol or methanol, e.g. methanol. The watermiscible organic solvent may be a C₂-C₄ nitrile, for exampleacetonitrile.

The hydrophilic vinylic monomer may comprise a generalised vinyl group,—CR═CH₂, where R is alkyl (e.g. where R is —CH₃ or —CH₂CH₃). Thehydrophilic vinylic monomer may comprise a vinyl group, —CH═CH₂. Thehydrophilic vinylic monomer may be vinylic monomers with a log P valueof less than about 1, e.g. a log P of less than about 0.5. The monomersmay be vinylic monomers with a log P of less than about 0.6. Themonomers may be vinylic monomers with a log P of less than about 0.5.For example, the monomers may be vinylic monomers with a log P of lessthan about 0.3 or of less than about 0.2, e.g. the monomers may bevinylic monomers with a log P of less than about 0.1. The monomers maybe vinylic monomers with a log P of less than about 0, e.g. with a log Pof less than about −0.2. The monomers may be vinylic monomers that havea log P of from 0.5 to −2, for example a log P of from 0 to −2, e.g. alog P of from −0.2 to −2. In particular, the monomers may be vinylicmonomers that also comprise a hydrophilic group, for example anacrylamide monomer or an acrylate monomer.

The monomer used in the method may be at least one compound of formula(I) where R¹ is —H, —CH₃ or —CH₂CH₃; R² is —OR³ or —N(R⁴)R⁵; R³ is —H,—C₁-C₆ alkyl, or —C₁-C₆ alcohol; and R⁴ and R⁵ are each independentlyselected from —H, —C₁-C₆ alkyl, —C₁-C₆ haloalkyl, —C₁-C₆ alcohol.

Where R³ is —C₁-C₆ alkyl or —C₁-C₆ alcohol, the alkyl or alcohol may besubstituted, where chemically possible, by 1 to 5 substituents (e.g. 1,2, 3 or 4) which are each independently at each occurrence selectedfrom: oxo, ═NR^(a), ═NOR^(a), halo, nitro, cyano, NR^(a)R^(a),NR^(a)S(O)₂R^(a), NR^(a)CONR^(a)R^(a), NR^(a)CO₂R^(a), OR^(a); SR^(a),S(O)R^(a), S(O)₂OR^(a), S(O)₂R^(a), S(O)₂NR^(a)R^(a), CO₂R^(a)C(O)R^(a), CONR^(a)R^(a), C₁-C₄-alkyl, C₂-C₄-alkenyl, C₂-C₄-alkynyl,C₁-C₄ haloalkyl; wherein R^(a) is independently at each occurrenceselected from: H and C₁-C₄ alkyl. For example, where R³ is —C₁-C₆ alkylor —C₁-C₆ alcohol, the alkyl or alcohol may be substituted, wherechemically possible, by 1 to 5 (e.g. 1, 2, 3 or 4) substituents whichare each independently at each occurrence selected from OR^(a) orCO₂R^(a), optionally where R^(a) is H.

Where R⁴ and/or R⁵ are —C₁-C₆ alkyl, —C₁-C₆ haloalkyl, —C₁-C₆ alcohol,each may be independently substituted, where chemically possible, by 1to 5 (e.g. 1, 2, 3 or 4) substituents which are each independently ateach occurrence selected from: oxo, ═NR^(a), ═NOR^(a), halo, nitro,cyano, NR^(a)R^(a), NR^(a)S(O)₂R^(a), NR^(a)CONR^(a)R^(a),NR^(a)CO₂R^(a), OR^(a); SR^(a), S(O)R^(a), S(O)₂OR^(a), S(O)₂R^(a),S(O)₂NR^(a)R^(a), CO₂R^(a) C(O)R^(a), CONR^(a)R^(a), C₁-C₄-alkyl,C₂-C₄-alkenyl, C₂-C₄-alkynyl, C₁-C₄ haloalkyl; wherein R^(a) isindependently at each occurrence selected from: H and C₁-C₄ alkyl. Forexample, where R⁴ and/or R⁵ are —C₁-C₆ alkyl, —C₁—C haloalkyl, —C₁-C₆alcohol, each may be independently substituted, where chemicallypossible, by 1 to 5 (e.g. 1, 2, 3 or 4) substituents which are eachindependently at each occurrence selected from OR^(a) or CO₂R^(a),optionally where R^(a) is H.

R¹ may be —H or —CH₃. For example, R¹ may be —H.

R² may be —OR³. R² may be —N(R⁴)R⁵.

R³ may be —H. R³ may be —C₁-C₆ alkyl. For example, R³ may be —C₁-C₆alkyl substituted by 1 or 2 substituents which are each independently ateach occurrence selected from: oxo, halo, cyano, NR^(a)R^(a),NR^(a)CONR^(a)R^(a), NR^(a)CO₂R^(a), OR^(a); CO₂R^(a) C(O)R^(a),CONR^(a)R^(a), C₁-C₄-alkyl, C₂-C₄-alkenyl, C₂-C₄-alkynyl, C₁-C₄haloalkyl; wherein R^(a) is independently at each occurrence selectedfrom: H and C₁-C₄ alkyl, for example wherein R^(a) is —H. R³ may be—C₁-C₆ alcohol. For example, R³ may be —C₁-C₆ alcohol substituted by 1or 2 substituents which are each independently at each occurrenceselected from: oxo, halo, cyano, NR^(a)R^(a), NR^(a)CONR^(a)R^(a),NR^(a)CO₂R^(a), OR^(a); CO₂R^(a) C(O)R^(a), CONR^(a)R^(a), C₁-C₄-alkyl,C₂-C₄-alkenyl, C₂-C₄-alkynyl, C₁-C₄ haloalkyl; wherein R^(a) isindependently at each occurrence selected from: H and C₁-C₄ alkyl, forexample wherein R^(a) is —H.

R⁴ may be —H or —C₁-C₆ alkyl. R⁴ may be —C₁-C₆ alkyl. For example, R⁴may be —C₁-C₆ alkyl substituted by 1 or 2 substituents which are eachindependently at each occurrence selected from: oxo, halo, cyano,NR^(a)R^(a), NR^(a)CONR^(a)R^(a), NR^(a)CO₂R^(a), OR^(a); CO₂R^(a)C(O)R^(a), CONR^(a)R^(a), C₁-C₄-alkyl, C₂-C₄-alkenyl, C₂-C₄-alkynyl,C₁-C₄ haloalkyl; wherein R^(a) is independently at each occurrenceselected from: H and C₁-C₄ alkyl, for example wherein R^(a) is —H.

R⁵ may be —H or —C₁-C₆ alkyl. R⁵ may be —C₁-C₆ alkyl. For example, R⁵may be —C₁-C₆ alkyl substituted by 1 or 2 substituents which are eachindependently at each occurrence selected from: oxo, halo, cyano,NR^(a)R^(a), NR^(a)CONR^(a)R^(a), NR^(a)CO₂R^(a), OR^(a); CO₂R^(a)C(O)R^(a), CONR^(a)R^(a), C₁-C₄-alkyl, C₂-C₄-alkenyl, C₂-C₄-alkynyl,C₁-C₄ haloalkyl; wherein R^(a) is independently at each occurrenceselected from: H and C₁-C₄ alkyl, for example wherein R^(a) is —H.

The compound of formula (I) may have a log P value of less than about 1,e.g. a log P of less than about 0.5. The compound of formula (I) mayhave a log P of less than about 0.6. The compound of formula (I) mayhave a log P of less than about 0.5. For example, the compound offormula (I) may have a log P of less than about 0.3 or of less thanabout 0.2, e.g. the compound of formula (I) may have a log P of lessthan about 0.1. The compound of formula (I) may have a log P of lessthan about 0, e.g. with a log P of less than about −0.2. The compound offormula (I) may have a log P of from 0.5 to −2, for example a log P offrom 0 to −2, e.g. a log P of from −0.2 to −2.

The monomer may comprise at least one hydrophilic vinylic monomercomprising a primary amide group (—C(O)NH₂).

Acrylamide monomers and/or acrylic monomers may be mentioned inparticular. Suitable monomers include acrylamide (prop-2-enamide),N-(hydroxymethyl)acrylamide, N-hydroxyethyl acrylamide,N-[tris(hydroxymethyl)methyl]acrylamide, 3-acrylamidopropanoic acid,methacrylamide, N-(2-hydroxyethyl)methacrylamide,N-(3-aminopropyl)methacrylamide, hydroxypropylacrylamide,N,N-dimethylacrylamide, 2-hydroxyethyl acrylate, 2-hydroxyethylmethacrylate, acrylic acid; and other acrylamide monomers, acrylicmonomers, methacrylamide monomers, or methacrylic monomers with a log Pvalue of less than about 1 (e.g. with a log P value of less than about0.5).

The solution (a) may comprise not more than 60% wt hydrophilic vinylicmonomer. For example, the solution (a) may comprise not more than 55% wtor 50% wt hydrophilic vinylic monomer. For example, the solution (a) maycomprise not more than 45% wt or 40% wt hydrophilic vinylic monomer;e.g. solution (a) may comprise not more than 30% wt hydrophilic vinylicmonomer. The solution (a) may comprise at least 2% wt hydrophilicvinylic monomer. The solution (a) may comprise at least 5% wthydrophilic vinylic monomer. The solution (a) may comprise at least 8%wt hydrophilic vinylic monomer. The solution (a) may comprise at least2% wt % (e.g. 5% wt) hydrophilic vinylic monomer and not more than 60%wt hydrophilic vinylic monomer, for example the solution (a) maycomprise at least 8% wt hydrophilic vinylic monomer and not more than60% wt hydrophilic vinylic monomer. The solution (a) may comprise atleast 2% wt % (e.g. 5% wt) hydrophilic vinylic monomer and not more than50% wt hydrophilic vinylic monomer, for example the solution (a) maycomprise at least 8% wt hydrophilic vinylic monomer and not more than50% wt hydrophilic vinylic monomer. The solution (a) may comprise atleast 2% wt % (e.g. 5% wt) hydrophilic vinylic monomer and not more than45% wt hydrophilic vinylic monomer, for example the solution (a) maycomprise at least 8% wt hydrophilic vinylic monomer and not more than45% wt hydrophilic vinylic monomer. The solution (a) may comprise atleast 2% wt % (e.g. 5% wt) hydrophilic vinylic monomer and not more than30% wt hydrophilic vinylic monomer, for example the solution (a) maycomprise at least 8% wt vinylic monomer and not more than 30% wthydrophilic vinylic monomer. The solution (a) may comprise at least 2%wt % (e.g. 5% wt) hydrophilic vinylic monomer and not more than 15%hydrophilic vinylic monomer, e.g. the solution (a) may comprise about10% wt hydrophilic vinylic monomer.

The hydrophilic vinylic monomer may comprise a mixture of monomers. Forexample, the monomer may comprise at least one monomer as defined aboveand at least one compatible functional monomer. A correspondingfunctional monomer is a hydrophilic vinylic monomer as defined herein,which comprises a carboxylic acid (—COOH), primary amine or secondaryamine. A functional monomer may be a vinylic monomer with a log P valueof less than about 1 (e.g. a log P of less than about 0.5), whichcomprises a carboxylic acid or primary amine. A functional monomer maybe a vinylic monomer with a log P of less than about 0.6. A functionalmonomer may be a vinylic monomer with a log P of less than about 0.5.For example, the functional monomer may be a vinylic monomer with a logP of less than about 0.3 or of less than about 0.2, e.g. the functionalmonomer may be a vinylic monomer with a log P of less than about 0.1. Afunctional monomer may be a vinylic monomer with a log P of less thanabout 0, e.g. with a log P of less than about −0.2. A functional monomermay be a vinylic monomer that has a log P of from 0.5 to −2, for examplea log P of from 0 to −2, e.g. a log P of from −0.2 to −2. A functionalmonomer may be a compound of formula (I) which comprises a carboxylicacid or primary amine. A functional monomer may be an acrylamide monomerwhich comprises a carboxylic acid or primary amine. Suitable functionalmonomers include 3-acrylamidopropionic acid, 4-acrylamidobutanoic acid,5-acrylamidopentanoic acid, N-(3-aminopropyl)methacrylamide, and acrylicacid.

Where at least one functional monomer is present, the amount of thefunctional monomer may be about 0.1 to about 100% mol, for example about0.2 to about 50% mol, e.g. about 0.5 to about 40% mol or about 1 toabout 30% mol (such as about 2 to about 20 mol %. The amount offunctional monomer may be about 0.1 to about 10% mol, for example about0.2 to about 5% mol, e.g. about 0.5 to about 2% mol. The % mol may referto the mol percent of the functional monomer included in the totalhydrophilic vinylic monomer of solution (a) (i.e. the solutioncomprising at least 2% wt hydrophilic vinylic monomer in water).

In highly crosslinked particles, the monomer may be or comprise acrosslinker. For example, the monomer may be or comprise at least onecompound of formula (IIa) or (IIb).

The crosslinker used in the method may comprise at least two (e.g. 2)vinyl groups (—CH═CH₂). The crosslinker used in the method may be atleast one compound of formula (IIa) or formula (IIb), wherein R⁶ isselected from —C₁-C₆ alkyl-, —C₁-C₆ heteroalkyl-, —C₁-C₆ cycloalkyl-,—C₁-C₀ hydroxyalkyl-, —C₁-C₀ ether-, or polyether comprising 2 to 100C₂-C₃ ether units; and R⁷ and R⁸ are each independently selected from—H, —C₁-C₆ alkyl, —C₁-C₆ heteroalkyl, —C₃-C₆ cycloalkyl, —C₁-C₆hydroxyalkyl, or —C₁-C₆ ether; R⁹ is —N(R¹¹)C(O)CH═CH₂; R¹⁰ is selectedfrom —H and —N(R¹²)C(O)CH═CH₂; and R¹¹ and R¹² are each independentlyselected from —H, —C₁-C₆ alkyl, —C₁-C₆ heteroalkyl, —C₃-C₆ cycloalkyl,—C₁-C₆ hydroxyalkyl, or —C₁-C₆ ether.

The crosslinker may be at least one compound of formula (IIa). Thecrosslinker may be at least one compound of formula (IIb).

R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹ and R¹² may be independently substituted, wherechemically possible, by 1 to 5 substituents which are each independentlyat each occurrence selected from: oxo, ═NR^(a), ═NOR^(a), halo, nitro,cyano, NR^(a)R^(a), NR^(a)S(O)₂R^(a), NR^(a)CONR^(a)R^(a),NR^(a)CO₂R^(a), OR^(a); SR^(a), S(O)R^(a), S(O)₂OR^(a), S(O)₂R^(a),S(O)₂NR^(a)R^(a), CO₂R^(a) C(O)R^(a), CONR^(a)R^(a), C₁-C₄-alkyl,C₂-C₄-alkenyl, C₂-C₄-alkynyl, C₁-C₄ haloalkyl; wherein R^(a) isindependently at each occurrence selected from: H, C₁-C₄ alkyl and C₁-C₄alkenyl.

R⁶ is selected from —C₁-C₆ alkyl-, —C₁-C₆ heteroalkyl-, —C₁-C₆cycloalkyl-, —C₁-C₆ hydroxyalkyl- and —C₁-C₆ ether-. R⁶ may be selectedfrom —C₁-C₆ alkyl- and —C₁-C₆ hydroxyalkyl-. R⁶ may be a —C₁-C₆ alkyl-,for example —CH₂—, —(CH₂)₂—, —(CH₂)₃—, or —(CH₂)₄—, e.g. —(CH₂)₂—. R⁶may be a —C₁-C₆ hydroxyalkyl-, for example —C(OH)H—, —(C(OH)H)₂—,—(C(OH)H)₃—, or —(C(OH)H)₄—, e.g. —(C(OH)H)₂—.

R⁶ may be a —C₁-C₆ heteroalkyl-, optionally wherein the heteroatom is anamine (e.g. a tertiary amine). For example a —C₁-C₆heteroalkyl-substituted by C(O)R^(a) on the hetero atom, optionallywherein the heteroatom is an amine, e.g. R⁶ may be—CH₂CH₂N(C(O)CH═CH₂)CH₂CH₂—.

Where R⁶ is a polyether, the polyether may be linear or branched. R⁶ maybe a polyether comprising 2 to 100 C₂-C₃ ether units, e.g. a polyethercomprising 2 to 50 C₂-C₃ ether units. R⁶ may be a polyether comprising 2to 100 C₂ ether units, e.g. a polyether comprising 2 to 50 C₂ etherunits. For example R⁶ may be —(CH₂)_(r)(OCH₂CH₂)_(n)O(CH₂)_(s), whereinr and s are each independently 2 or 3 (e.g. 2); and n is an integer from1 to 100 (e.g. 5 to 50 or 5 to 25). Without wishing to be bound by anytheory, it is believed that crosslinkers comprising a polyether (e.g.where R⁶ is a polyether) have excellent solubility in the aqueous phase.This means that, while such crosslinkers may be used to provideparticles with a low level of crosslinking (e.g. 1-20% wt crosslinker,or 1-10% wt crosslinker), such polyether comprising crosslinkers areparticularly suitable for providing particles comprising relatively highlevels of crosslinking, for example >20% wt crosslinker, >25% wtcrosslinker, or >30% wt crosslinker. For example, the level ofcrosslinking may be 10-90% wt crosslinker, 20-80% wt crosslinker or25-75% wt crosslinker, e.g. 25-60% wt crosslinker or 30-50% wtcrosslinker.

R⁷ and/or R⁸ and/or R¹¹ and/or R¹² may be H. For example, R⁷ and/or R⁸may be H. For example, R¹¹ and/or R¹² may be H.

The compound of formula (II) may have a log P value of less than about1, e.g. a log P of less than about 0.5. The compound of formula (II) mayhave a log P of less than about 0.6. The compound of formula (II) mayhave a log P of less than about 0.5. For example, the compound offormula (II) may have a log P of less than about 0.3 or of less thanabout 0.2, e.g. the compound of formula (II) may have a log P of lessthan about 0.1. The compound of formula (II) may have a log P of lessthan about 0, e.g. with a log P of less than about −0.2. The compound offormula (II) may have a log P of from 0.5 to −2, for example a log P offrom 0 to −2, e.g. a log P of from −0.2 to −2.

Exemplary crosslinkers of use in embodiments of the method includeN,N′-(1,2-dihydroxyethylene)bisacrylamide,N,N′-methylenebis(acrylamide), N,N′-ethylenebis(acrylamide), piperazinediacrylamide, glycerol 1,3-diglycerolate diacrylate;N,N′-((ethane-1,2-diylbis(oxy))bis(ethane-2,1-diyl))diacrylamide,polyethyleneglycol diacrylamide (MW≤2000), 4-arm PEG-acrylamide(MW≤2000), N,N-bis(2-acrylamidoethyl)acrylamide. The exemplarycrosslinkersN,N′-((ethane-1,2-diylbis(oxy))bis(ethane-2,1-diyl))diacrylamide,polyethyleneglycol diacrylamide (MW≤2000) and 4-Arm PEG-Acrylamide(MW≤2000) (e.g.,N′-((ethane-1,2-diylbis(oxy))bis(ethane-2,1-diyl))diacrylamide) areparticular suitable for use in highly crosslinked particles andmechanically more stable particles, i.e. particles with a level ofcrosslinking of at least 20% wt crosslinker (e.g. a level ofcrosslinking of at least 30% wt crosslinker). Embodiments may alsoinclude the use of a combination of crosslinkers.

The crosslinker may be a compound that does not comprise a primaryamine, secondary amine, hydroxy or carboxylic acid. The crosslinker maybe a compound of formula (IIa) or formula (IIb) that does not comprise aprimary amine, secondary amine, hydroxyl or carboxylic acid

The level of crosslinking in the crosslinked hydrogel polymer particlesformed by the method can be expressed as the percentage by weight (% wt)of crosslinker monomer included in the total monomer used in thepolymerisation. Typical levels of crosslinking include >5% wtcrosslinker, for example >10% wt crosslinker, or >15% wt crosslinker,e.g. >20% wt crosslinker. The level of crosslinking may also be, forinstance 5-60% wt crosslinker, for example 10-50% wt crosslinker. Thelevel of crosslinking may also be, for instance 15-40% wt crosslinker,for example 20-40% wt crosslinker, e.g. 20-30% wt crosslinker. Thesolution (a) may therefore comprise at an amount of crosslinker in % wtthat corresponds to 5-60% of the amount of hydrophilic vinylic monomer,for example 10-50% of the amount of hydrophilic vinylic monomer. Thesolution (a) may therefore comprise at an amount of crosslinker in % wtthat corresponds to, for instance 15-40% of the amount of hydrophilicvinylic monomer, for example 20-40% of the amount of hydrophilic vinylicmonomer, e.g. 20-30% of the amount of hydrophilic vinylic monomer.

The level of crosslinking may be >30% wt crosslinker or >40% wtcrosslinker (e.g. >50% wt crosslinker), for example in highlycrosslinked particles. The level of crosslinking may be 10-90% wtcrosslinker, 20-80% wt crosslinker or 25-75% wt crosslinker, e.g. 25-60%wt crosslinker or 30-50% wt crosslinker. In highly crosslinked particlesthe level of crosslinking may be up to 100% wt crosslinker, for examplethe hydrophilic vinylic monomer may be a crosslinker, e.g. thehydrophilic vinyilic monomer and the crosslinker may be the samecompound. The solution (a) may therefore comprise at an amount ofcrosslinker in % wt that corresponds to 20-80% of the amount ofhydrophilic vinylic monomer, for example 25-60% of the amount ofhydrophilic vinylic monomer.

As a particular hydrophilic vinylic monomer may be mentioned acrylamide(prop-2-enamide), for which 1,2-dihydroxy bisacrylamide is a suitablecrosslinker. As a particular hydrophilic vinylic monomer may bementioned hydroxymethyl acrylamide, for whichN,N′-((ethane-1,2-diylbis(oxy))bis(ethane-2,1-diyl))diacrylamide is asuitable crosslinker. As a particular hydrophilic vinylic monomer may bementioned hydroxyethyl acrylamide, for whichN,N′-((ethane-1,2-diylbis(oxy))bis(ethane-2,1-diyl))diacrylamide is asuitable crosslinker.

The stabilizer may be or comprise a non-ionic surfactant, for example anon-ionic polymeric surfactant. The non-ionic surfactant may comprise atleast one oligomeric surfactant. For example, the non-ionic surfactantmay comprise an oligomeric surfactant. The non-ionic polymeric oroligomeric surfactant may comprise at least one polyethyleneoxy group orat least one polypropyleneoxy group. The non-ionic polymeric surfactantmay comprise at least one polyethyleneoxy group. The non-ionicoligomeric surfactant may comprise at least one polyethyleneoxy group orat least one polypropyleneoxy group. The stabiliser may be selected fromor comprise hypermer 2296, Abil WE09, Abil EM90 and sorbitane monooleate(Span 80).

Polymerisation may comprise activating the radical initiator. Theradical initiator is typically activated by heating to form radicalsthat will initiate the polymerization reaction, however other methods ofactivation may be used, e.g. radiation. Activating the radical initiatormay comprise heating the emulsion comprising swollen particles. Theheating typically comprises heating the emulsion to a temperature abovethe temperature at which the step of allowing the monodisperse seedparticles to form swollen particles was performed. The heating maycomprise heating to a temperature of at least 40° C., for exampleheating to a temperature of at least 50° C., e.g. a temperature of atleast 60° C. or a temperature of at least 70° C.

The radical initiator may be or comprise a peroxide-initiator or anazo-initiator. For example, the radical initiator may be aperoxide-initiator. For example, the radical initiator may be anazo-initiator. An exemplary radical initiator is2,2′-azobis-2,4-dimethyl valeronitrile. An exemplary radical initiatoris 2,2′-azodi(2-methylbutyronitrile). The radical initiator may bepresent in solution (a). The radical initiator may be present insolution (b). The radical initiator may be present in an amount of fromabout 0.1 wt % to about 1.5 wt % in the emulsion. For example theradical initiator may be present in the emulsion in an amount of fromabout 0.6 wt % to about 1.2 wt %; e.g. the radical initiator may bepresent in the emulsion in an amount of about 0.8% in the emulsion.

The organic solvent used in the methods of forming monodispersecrosslinked hydrogel polymer particles may comprise (or consist of) atleast one of an aliphatic hydrocarbon, an aliphatic carbonate, analiphatic ester, an aliphatic ether, an aromatic hydrocarbon, or asilicone, or a combination thereof. For example, the organic solvent maycomprise (or consist of) at least two of an aliphatic hydrocarbon, analiphatic carbonate, an aliphatic ester, an aliphatic ether, an aromatichydrocarbon, and a silicone; or the organic solvent may comprise (orconsist of) at least three of an aliphatic hydrocarbon, an aliphaticcarbonate, an aliphatic ester, an aliphatic ether, an aromatichydrocarbon, and a silicone. The organic solvent may comprise (orconsist of) a mixture of heptane and toluene. The organic solvent maycomprise (or consist of) a mixture of aliphatic hydrocarbons. Theorganic solvent may comprise (or consist of) bis(2-ethylhexyl)carbonate. The organic solvent may comprise (or consist of)bis(2-ethylhexyl) carbonate, aliphatic hydrocarbons and aromatichydrocarbons. The organic solvent may comprise (or consist of)bis(2-ethylhexyl)adipate.

The monodisperse seed particles added to the water-in-oil emulsion (c)may have the characteristics of monodisperse seed particles formedaccording to a method of preparing monodisperse seed particles disclosedherein. The monodisperse seed particles added to water-in-oil emulsion(c) may be monodisperse seed particles formed according to a method ofpreparing monodisperse seed particles disclosed herein. The monodisperseseed particles may be monodisperse seed particles of the disclosure,e.g. monodisperse seed particles of the invention.

In an embodiment the present invention provides a method of formingmonodisperse crosslinked hydrogel polymer particles. The methodcomprises forming a solution (a) of at least 2% wt of a hydrophilicvinylic monomer in an aqueous solution, the aqueous solution alsocomprising a chain transfer agent; forming a solution (b) of stabilizerin an organic solvent, wherein the organic solvent is not miscible inwater, and wherein at least one of solution (a) and solution (b)comprises a radical initiator; mixing solutions (a) and (b) to form awater-in-oil emulsion (c) and adding monodisperse seed particles to theemulsion; allowing the monodisperse seed particles to form swollenparticles in the emulsion; polymerising the swollen particles to formmonodisperse polymer particles; forming a solution (d) of stabilizer inan organic solvent, wherein the organic solvent is not miscible inwater; forming a solution (e) of at least 2% wt of a hydrophilic vinylicmonomer in an aqueous solution, the aqueous solution also comprising acrosslinker comprising at least two vinyl groups, wherein at least oneof solution (d) and solution (e) comprises a radical initiator; mixingsolutions (d) and (e) to form a water-in-oil emulsion (f) and adding themonodisperse polymer particles to the emulsion; allowing themonodisperse polymer particles to form swollen polymer particles in theemulsion; and polymerising the swollen polymer particles to form themonodisperse crosslinked hydrogel polymer particles. As explained below,this method may be considered a two-stage method, as it comprises afirst swelling step and first polymerisation step, followed by a secondswelling step and a second polymerisation step. Compared to a singlestage-method, the two-stage method may provide large monodispersecrosslinked hydrogel crosslinked polymer particles.

Similarly to the single-stage method, the two-stage method involvesswelling the seed particles with an aqueous discontinuous phase in anoil continuous phase. A schematic of this process, which may beconsidered a two-stage process, as it comprises a two steps of swellingand two steps of polymerisation, is provided in FIG. 3 (illustrated withspecific monomers) and FIG. 4 (illustrated more generally). The methodillustrated in FIGS. 3 and 4 is conveniently divided into 4 steps:swelling of the monodisperse seed particles 100 with monomer 103 andchain transfer agent 105 to form swollen (monodisperse) seed particles110; polymerisation of the monomer in the swollen seed particles to form(non-crosslinked) hydrophilic polymer particles 130; swelling of thehydrophilic polymer particles with monomer 103 and cross-linker 102 toform swollen hydrophilic polymer particles 140; and polymerisation ofmonomer in the hydrogel polymer particles to form crosslinked hydrogelpolymer particles 150. Prior to the first step a water-in-oil emulsionis formed. The water-in-oil emulsion is formed by mixing an aqueoussolution comprising a monomer, e.g. hydroxymethylacrylamide 103, and achain transfer agent, e.g. 1-thioglycerol 105, with an oil phasecomprising a steric stabiliser, with the emulsion formed when the waterand oil phases are agitated (e.g. by stirring). The water-in-oilemulsion also typically contains an initiator, which may have been addedto the oil phase and/or the aqueous phase. The initiator is a compoundthat upon activation will initiate polymerisation of the monomer. Themonodisperse seed particles 100 comprise non-crosslinked oligomers ofpoly N,N-dimethylacrylamide 131 and are considered “activated seedparticles”. The seed particles 100 may be prepared as describedelsewhere in this application.

In the first step monodisperse seed particles 100 are added to thewater-in-oil emulsion and the emulsion is agitated for a period of time(for instance for at least 30 minutes or for at least 1 hour, typicallyfor 4-48 hours). During this period of time the monomer 103 and chaintransfer agent 105 diffuse into the activated seed particles 100 to formthe swollen seed particles 110. The swollen seed particles 110 comprisea mixture of at least the monomer 103, the chain transfer agent 105 andpolymer from the activated seed particles 100. The swollen seedparticles 110 may also include other components, for instance one ormore porogens, which can enter the particles if included in thewater-in-oil emulsion. For example, in the illustrated method, water isalso present in the swollen seed particles 110, and this water may beconsidered a porogen. The second step comprises polymerisation of themonomer 103 to form the (non-crosslinked) hydrophilic polymer particles130. In the second step polymerisation is initiated by activating theinitiator, for example by heating the emulsion. The presence of thechain transfer agent 105 results in relatively short polymers ofpolyhydroxymethyl acrylamide 132 (e.g. oligomers) in the hydrogelpolymer particles. These relatively short polymers, combined with thelack of cross-linking, means that the hydrophilic polymer particles 130may act as larger “activated seed particles” suitable for a furtherround of swelling and polymerisation. The hydrophilic polymer particleswill contain the non-crosslinked relatively short polymers of polymersof polyhydroxymethyl acrylamide 132, as well as a (typically smallerproportion) of non-crosslinked oligomers of poly N,N-dimethylacrylamide131.

Prior to the third step a water-in-oil emulsion is formed. Thewater-in-oil emulsion is formed by mixing an aqueous solution comprisinga monomer, e.g. hydroxymethylacrylamide 103, and a crosslinker, e.g.1,2-dihydroxy bisacrylamide 102 with an oil phase comprising a stericstabiliser. If a (very) highly crosslinked particle is desired, it isalso possible to replace the monomer with a crosslinker, so that theonly monomers present in the water-in-oil emulsion are crosslinkingmonomers (i.e. crosslinkers). The water-in-oil emulsion also typicallycontains an initiator, which may have been added to either the oil phaseor the aqueous phase. The initiator is a compound that upon activationwill initiate polymerisation of the monomer and the crosslinker. In thethird step (second round of swelling) hydrophilic polymer particles 130are added to the water-in-oil emulsion and the emulsion is agitated fora period of time (for instance for at least 30 minutes or for at least 1hour, typically for 4-48 hours). During this period of time the monomer103 and crosslinker 102 diffuse into the hydrophilic polymer particles130 to form the swollen hydrogel polymer particles 140. The swollenhydrophilic polymer particles 140 comprise a mixture of at least themonomer 103, crosslinker 102 and polymers from the hydrophilic polymerparticles 130. The swollen hydrogel polymer particles 140 may alsoinclude other components, for instance one or more porogens, which canenter the particles if included in the water-in-oil emulsion. Forexample, in the illustrated method, water is also present in the swollenseed particles 110, and this water may be considered a porogen. Thefourth step comprises polymerisation of the monomer 103 and crosslinker102 to form the crosslinked hydrogel polymer particles 104. In thefourth step polymerisation is initiated by activating the initiator, forexample by heating the emulsion.

The monomer, chain transfer agent, radical initiator, stabiliser,cross-linker, organic solvent and optional porogen may each be asdefined elsewhere herein. The monomer may be or comprisehydroxymethylacrylamide or hydroxyethylacrylamide. The chain transferagent may be or comprise 1-thioglycerol. The radical initiator may be orcomprise azobisdimethyl valeronitrile, e.g.2,2′-azodi(2-methylbutyronitrile) (AMBN). The stabiliser may be orcomprise Hypermer 2296, Abil WE09, and Abil EM90. The crosslinker may be1,2-dihydroxy bisacrylamide orN,N′-((ethane-1,2-diylbis(oxy))bis(ethane-2,1-diyl))diacrylamide.

The solution (a) may comprise not more than 60% wt hydrophilic vinylicmonomer. For example, the solution (a) may comprise not more than 55% wtor 50% wt hydrophilic vinylic monomer. For example, the solution (a) maycomprise not more than 45% wt or 40% wt hydrophilic vinylic monomer;e.g. solution (a) may comprise not more than 30% wt hydrophilic vinylicmonomer. The solution (a) may comprise at least 2% wt hydrophilicvinylic monomer. The solution (a) may comprise at least 5% wthydrophilic vinylic monomer. The solution (a) may comprise at least 8%wt hydrophilic vinylic monomer. The solution (a) may comprise at least2% wt % (e.g. 5% wt) hydrophilic vinylic monomer and not more than 60%wt hydrophilic vinylic monomer, for example the solution (a) maycomprise at least 8% wt hydrophilic vinylic monomer and not more than60% wt hydrophilic vinylic monomer. The solution (a) may comprise atleast 2% wt % (e.g. 5% wt) hydrophilic vinylic monomer and not more than50% wt hydrophilic vinylic monomer, for example the solution (a) maycomprise at least 8% wt hydrophilic vinylic monomer and not more than50% wt hydrophilic vinylic monomer. The solution (a) may comprise atleast 2% wt % (e.g. 5% wt) hydrophilic vinylic monomer and not more than45% wt hydrophilic vinylic monomer, for example the solution (a) maycomprise at least 8% wt hydrophilic vinylic monomer and not more than45% wt hydrophilic vinylic monomer. The solution (a) may comprise atleast 2% wt % (e.g. 5% wt) hydrophilic vinylic monomer and not more than30% wt hydrophilic vinylic monomer, for example the solution (a) maycomprise at least 8% wt vinylic monomer and not more than 30% wthydrophilic vinylic monomer. The solution (a) may comprise at least 2%wt % (e.g. 5% wt) hydrophilic vinylic monomer and not more than 15%hydrophilic vinylic monomer, e.g. the solution (a) may comprise about10% wt hydrophilic vinylic monomer.

The solution (e) may comprise not more than 60% wt hydrophilic vinylicmonomer. For example, the solution (e) may comprise not more than 55% wtor 50% wt hydrophilic vinylic monomer. For example, the solution (e) maycomprise not more than 45% wt or 40% wt hydrophilic vinylic monomer;e.g. solution (e) may comprise not more than 30% wt hydrophilic vinylicmonomer. The solution (e) may comprise at least 2% wt hydrophilicvinylic monomer. The solution (e) may comprise at least 5% wthydrophilic vinylic monomer. The solution (e) may comprise at least 8%wt hydrophilic vinylic monomer. The solution (e) may comprise at least2% wt % (e.g. 5% wt) hydrophilic vinylic monomer and not more than 60%wt hydrophilic vinylic monomer, for example the solution (e) maycomprise at least 8% wt hydrophilic vinylic monomer and not more than60% wt hydrophilic vinylic monomer. The solution (e) may comprise atleast 2% wt % (e.g. 5% wt) hydrophilic vinylic monomer and not more than50% wt hydrophilic vinylic monomer, for example the solution (e) maycomprise at least 8% wt hydrophilic vinylic monomer and not more than50% wt hydrophilic vinylic monomer. The solution (e) may comprise atleast 2% wt % (e.g. 5% wt) hydrophilic vinylic monomer and not more than45% wt hydrophilic vinylic monomer, for example the solution (e) maycomprise at least 8% wt hydrophilic vinylic monomer and not more than45% wt hydrophilic vinylic monomer. The solution (e) may comprise atleast 2% wt % (e.g. 5% wt) hydrophilic vinylic monomer and not more than30% wt hydrophilic vinylic monomer, for example the solution (e) maycomprise at least 8% wt vinylic monomer and not more than 30% wthydrophilic vinylic monomer. The solution (e) may comprise at least 2%wt % (e.g. 5% wt) hydrophilic vinylic monomer and not more than 15%hydrophilic vinylic monomer, e.g. the solution (e) may comprise about10% wt hydrophilic vinylic monomer.

In highly crosslinked particles, the hydrophilic vinylic monomer ofsolution (e) may be or comprise a crosslinker. For example, the monomermay be or comprise at least one compound of formula (IIa) or (IIb).

The level of crosslinking in the crosslinked hydrogel polymer particlesformed by the method can be expressed as the percentage by weight (% wt)of crosslinker monomer included in the total monomer used in thepolymerisation (i.e. in the second polymerisation step). The % wt ofcrosslinker monomer may be equivalent to the % wt of the crosslinker inmatrix polymer (i.e. the % wt of crosslinker in the dry weight of thecrosslinked polymer particles). Typical levels of crosslinkinginclude >5% wt crosslinker, for example >10% wt crosslinker, or >15% wtcrosslinker, e.g. >20% wt crosslinker (such as >30% wt crosslinker). Thelevel of crosslinking may also be, for instance 5-60% wt crosslinker,for example 10-50% wt crosslinker. The level of crosslinking may alsobe, for instance 15-40% wt crosslinker, for example 20-40% wtcrosslinker, e.g. 20-30% wt crosslinker. The level of crosslinking maybe 20-80% wt crosslinker or 25-75% wt crosslinker, e.g. 25-60% wtcrosslinker or 30-50% wt crosslinker; for example in high densityparticles. The solution (e) may therefore comprise at an amount ofcrosslinker in % wt that corresponds to 5-60% of the amount ofhydrophilic vinylic monomer, for example 10-50% of the amount ofhydrophilic vinylic monomer. The solution (e) may therefore comprise atan amount of crosslinker in % wt that corresponds to, for instance15-40% of the amount of hydrophilic vinylic monomer, for example 20-40%of the amount of hydrophilic vinylic monomer, e.g. 20-30% of the amountof hydrophilic vinylic monomer.

The level of crosslinking may be >30% wt crosslinker or >40% wtcrosslinker (e.g. >50% wt crosslinker), for example in highlycrosslinked particles. The level of crosslinking may be 10-90% wtcrosslinker, 20-80% wt crosslinker or 25-75% wt crosslinker, e.g. 25-60%wt crosslinker or 30-50% wt crosslinker. In highly crosslinked particlesthe level of crosslinking may be up to 100% wt crosslinker, for examplethe hydrophilic vinylic monomer in solution (e) may be a crosslinker,e.g. the hydrophilic vinyilic monomer and the crosslinker may be thesame compound. The solution (e) may comprise at an amount of crosslinkerin % wt that corresponds to 20-80% of the amount of hydrophilic vinylicmonomer, for example 25-60% of the amount of hydrophilic vinylicmonomer.

The radical initiator may be present in an amount of from about 0.1 wt %to about 1.5 wt % in each or at least one emulsion. For example theradical initiator may be present in each or at least one emulsion in anamount of from about 0.6 wt % to about 1.2 wt %; e.g. the radicalinitiator may be present in each or at least one emulsion in an amountof about 0.8 wt % in each or at least one emulsion.

The addition of a chain transfer agent reduces the molecular weight ofthe polymer of the monodisperse polymer particles by reacting with thefree radical of a growing polymer chain to terminate the chain andtransfer the lone electron to a radical species derived from the chaintransfer agent. The radical species derived from the chain transferagent may then react with a monomer to form a radical from the monomer,which can then react with another monomer to commence formation of a newpolymer chain. The chain transfer reagent may be a thiol or ahaloalkane. For example, the chain transfer agent may be selected fromthiols (e.g. 1-thioglycerol, 1-octanethiol, hexane thiol,6-mercapto-1-hexanol, benzylthiol), alkyl thiols (e.g. 1-octanethiol,hexane thiol) and thipolyols (e.g. 1-thioglycerol). The chain transferagent may be or comprise 1-thioglycerol. The total amount of chaintransfer agent added can be in the range 1 mol per 10 mol of monomer to1 mol per 300 mol of monomer, for example 1 mol per 20 mol of monomer to1 mol per 100 mol monomer, e.g. approximately 1 mol chain transfer agentper 30 mol of monomer.

The monodisperse crosslinked hydrogel polymer particles formed accordingto a method of the invention may be porous. For example, themonodisperse crosslinked hydrogel polymer particles formed may have aporosity of at least 5%, e.g. at least 10%. The disclosure includesporous monodisperse crosslinked hydrogel polymer particles having aporosity of from 20% to 95%, particularly of from 30% to 90%, e.g. offrom 40% to 90%, such as from 50% to 80%. The porous monodispersecrosslinked hydrogel polymer particles formed may be transparent tosolvated molecules, for example the porous monodisperse crosslinkedhydrogel polymer particles may be transparent to oligonucleotides andnucleic acid amplification reagents and sequencing reagents (e.g.primers, nucleotides and polymerases).

The monodisperse crosslinked hydrogel polymer particles formed accordingto a method of the invention may have a mode diameter of from 0.5 μm to10 μm, for example of from 0.5 μm to 5 μm. The monodisperse crosslinkedhydrogel polymer particles formed may have a mode diameter of at least500 nm, e.g. at least 600 nm, optionally at least 800 nm, as in the caseof particles having a diameter of at least 1 μm. The monodispersecrosslinked hydrogel polymer particles formed according to a method ofthe invention may have a mode diameter of not more than 10 μm, e.g. notmore than 5 μm, optionally not more than 3 μm, as in the case ofparticles having a diameter of not more than 2 μm. The size and sizedistribution of the monodisperse crosslinked hydrogel polymer particlesformed according to a method of the invention may be determined asdescribed below under the heading “analytical methods”.

The monodisperse crosslinked hydrogel polymer particles formed accordingto a method of the invention may have a CV of less than 20%, for exampleless than 15%. For example, the particles may have a CV of less than10%, such as a CV of less than 8%, e.g. a CV of less than 5%.

The monodisperse crosslinked hydrogel polymer particles formed accordingto a method of the invention may comprise functional groups. Thefunctional groups may be selected from a hydroxyl, a carboxylic acid(—COOH), a primary amine or a secondary amine. In an embodiment, thefunctional groups are provided by a hydrophilic vinylic monomer (e.g.compound of formula (I)) and not a crosslinker (e.g. compound of formula(IIa) or formula (IIb)). The functional groups may be enhanced tofacilitate binding with target analytes (e.g. oligonucleotides) ortarget receptors. Exemplary methods of enhancing functional groups ofthe particles are described herein in the section relating to “Uses ofthe Particles”.

The monodisperse crosslinked hydrogel polymer particles formed accordingto methods of the invention may comprise oligonucleotides attached tothe particles. The oligonucleotides may be attached to the monodispersecrosslinked hydrogel polymer particles via a linker. For example, eachof (or a proportion of) the monodisperse hydrogel polymer particles maycomprise a plurality of oligonucleotides attached to the particle. Theplurality of oligonucleotides may be identical for each individualcrosslinked hydrogel polymer particle. For example, a first crosslinkedhydrogel polymer particle may comprise a plurality of oligonucleotideshaving a first sequence attached to the first particle, and a secondcrosslinked hydrogel polymer particle may comprise a plurality ofoligonucleotides having a second sequence attached to the secondcrosslinked hydrogel polymer particle. Where the monodispersecrosslinked hydrogel polymer particles are porous, the oligonucleotidesmay be attached to the outer surface of the particle or attached insidea pore. The pores may be of sufficient size to render the particletransparent to the oligonucleotides, such that the oliogonucleotides maybe located partly or wholly within the pores, even when a polymerase isattached to the oligonucleotide.

The monodisperse crosslinked hydrogel polymer particles formed accordingto a method of the invention may be stable (i.e. resistant to polymerdegradation) in aqueous solution over the typical temperature ranges ofaqueous solutions. The monodisperse crosslinked hydrogel polymerparticles formed may be stable at a temperature of up to about 100° C.For example the monodisperse crosslinked hydrogel polymer particlesformed may be stable in a temperature range of from about 0° C. to about100° C.

An embodiment of the invention provides particles obtained by, or havingthe characteristics of particles obtained by, the preparative processesdescribed herein.

The polymer particle forming processes described herein may be worked tobe highly reproducible and scaleable. The invention may therefore enableconsistency between and within batches, which is a prerequisite forindustrial application. The invention may also enable production ofpilot scale batches of e.g. at least 300 g as well as kilogram scaleindustrial batches.

The polymer particle forming processes described herein can be performedconsistently without the problems which in practice can arise withemulsion polymerisation, e.g. agglomeration of particles as well asvariation in the product.

Uses of the Particles

The particles can be used in many applications, e.g. polynucleotidesequencing, polynucleotide sequencing, information storage, colorimaging, bioprocessing, diagnostic microbiology, biosensors and drugdelivery.

The polymer particles can be activated to facilitate conjugation with atarget analyte, such as a polynucleotide. In addition to the methodsdescribed below, suitable methods of activation and bioconjugation aredescribed in G. T. Hermanson, Bioconjugate Techniques, 2013 (3^(rd)Edition), Academic Press, the content of which is incorporated byreference herein in its entirety.

For example, functional groups on the polymeric particle can be enhancedto permit binding with target analytes or analyte receptors. In aparticular example, functional groups of the polymer particles can bemodified with reagents capable of converting the functional groups toreactive moieties that can undergo nucleophilic or electrophilicsubstitution. For example, hydroxyl groups on the polymer particles canbe activated by replacing at least a portion of the hydroxyl groups witha sulfonate group or chlorine. Exemplary sulfonate groups can be derivedfrom tresyl, mesyl, tosyl, or tosyl chloride, or any combinationthereof. Sulfonate can act to permit nucleophiles to replace thesulfonate. The sulfonate may further react with liberated chlorine toprovide a chlorinated groups that can be used in a process to conjugatethe particles. In another example, where the polymer particles compriseamine groups or carboxylic acid groups (e.g. from a functional monomer),the amine groups or carboxylic acid groups can be activated.

For example, target analyte or analyte receptors can bind to the polymerparticles through nucleophilic substitution with the sulfonate group orother activated group. In particular example, target analyte receptorsterminated with a nucleophile, such as an amine or a thiol, can undergonucleophilic substitution to replace the sulfonate groups on the surfaceof the polymer particles. As a result of the activation, conjugatedparticles can be formed.

In another example, sulfonated particles can be further reacted withmono- or multi-functional mono- or multi-nucleophilic reagents that canform an attachment to the particle while maintaining nucleophilicactivity for oligonucleotides comprising electrophilic groups, such asmaleimide. In addition, the residual nucleophilic activity can beconverted to electrophilic activity by attachment to reagents comprisingmulti-electrophilic groups, which are subsequently to attach tooligonucleotides comprising nucleophilic groups.

In another example, a monomer containing the functional group(functional monomer) may be included in the mixture of monomers duringthe polymerization. The functional monomer can include, for example, anacrylamide containing a carboxylic acid, ester, halogen or other aminereactive group. The ester group may be hydrolyzed before the reactionwith an amine oligonucleotide.

Other activation chemistries include incorporating multiple steps toconvert a specified functional group to accommodate specific desiredlinkages. For example, the sulfonate modified hydroxyl group can beconverted into a nucleophilic group through several methods. In anexample, reaction of the sulfonate with azide anion yields an azidesubstituted hydrophilic polymer. The azide can be used directly toconjugate to an acetylene substituted biomolecule via “CLICK” chemistrythat can be performed with or without copper catalysis. Optionally, theazide can be converted to amine by, for example, catalytic reductionwith hydrogen or reduction with an organic phosphine. The resultingamine can then be converted to an electrophilic group with a variety ofreagents, such as di-isocyanates, bis-NHS esters, cyanuric chloride, ora combination thereof. In an example, using di-isocyanates yields a urealinkage between the polymer and a linker that results in a residualisocyanate group that is capable of reacting with an amino substitutedbiomolecule to yield a urea linkage between the linker and thebiomolecule. In another example, using bis-NHS esters yields an amidelinkage between the polymer and the linker and a residual NHS estergroup that is capable of reacting with an amino substituted biomoleculeto yield an amide linkage between the linker and the biomolecule. In afurther example, using cyanuric chloride yields an amino-triazinelinkage between the polymer and the linker and two residualchloro-triazine groups one of which is capable of reacting with an aminosubstituted biomolecule to yield an amino-triazine linkage between thelinker and the biomolecule. Other nucleophilic groups can beincorporated into the particle via sulfonate activation. For example,reaction of sulfonated particles with thiobenzoic acid anion andhydrolysis of the consequent thiobenzoate incorporates a thiol into theparticle which can be subsequently reacted with a maleimide substitutedbiomolecule to yield a thio-succinimide linkage to the biomolecule.Thiol can also be reacted with a bromo-acetyl group.

Covalent linkages of biomolecules onto refractory or polymericsubstrates can be created using electrophilic moieties on the substratecoupled with nucleophilic moieties on the biomolecule or nucleophiliclinkages on the substrate coupled with electrophilic linkages on thebiomolecule. Because of the hydrophilic nature of most commonbiomolecules of interest, the solvent of choice for these couplings iswater or water containing some water soluble organic solvent in order todisperse the biomolecule onto the substrate. In particular,polynucleotides are generally coupled to substrates in water systemsbecause of their poly-anionic nature. Because water competes with thenucleophile for the electrophile by hydrolyzing the electrophile to aninactive moiety for conjugation, aqueous systems generally result in lowyields of coupled product, where the yield is based on the electrophilicportion of the couple. When high yields of electrophilic portion of thereaction couple are desired, high concentrations of the nucleophile arerequired to drive the reaction and mitigate hydrolysis, resulting ininefficient use of the nucleophile. In the case of polynucleic acids,the metal counter ion of the phosphate can be replaced with a lipophiliccounter-ion, in order to help solubilize the biomolecule in polar,non-reactive, non-aqueous solvents. These solvents can include amides orureas such as formamide, N,N-dimethylformamide, acetamide,N,N-dimethylacetamide, hexamethylphosphoramide, pyrrolidone,N-methylpyrrolidone, N,N,N′,N′-tetramethylurea,N,N′-dimethyl-N,N′-trimethyleneurea, or a combination thereof;carbonates such as dimethyl carbonate, propylene carbonate, or acombination thereof; ethers such as tetrahydrofuran; sulfoxides andsulfones such as dimethylsulfoxide, dimethylsulfone, or a combinationthereof; hindered alcohols such as tert-butyl alcohol; or a combinationthereof. Lipophilic cations can include tetraalkylammomiun ortetraarylammonium cations such as tetramethylamonium, tetraethylamonium,tetrapropylamonium, tetrabutylamonium, tetrapentylamonium,tetrahexylamonium, tetraheptylamonium, tetraoctylamonium, and alkyl andaryl mixtures thereof, tetraarylphosphonium cations such astetraphenylphosphonium, tetraalkylarsonium or tetraarylarsonium such astetraphenylarsonium, and trialkylsulfonium cations such astrimethylsulfonium, or a combination thereof. The conversion ofpolynucleic acids into organic solvent soluble materials by exchangingmetal cations with lipophilic cations can be performed by a variety ofstandard cation exchange techniques.

The polymer particles can be activated to facilitate conjugation with atarget analyte, such as a polynucleotide. For example, functional groupson the crosslinked hydrogel polymer particles can be enhanced to permitbinding with target analytes or analyte receptors. In a particularexample, functional groups of the polymer can be modified with reagentscapable of converting hydrophilic polymer functional groups to reactivemoieties that can undergo nucleophilic or electrophilic substitution. Inparticular, where the polymer particles have carboxyl functionality,these can be activated to facilitate conjugation, for example tobiomolecules, such as nucleic acids.

In embodiments where the particles are formed with a comonomer includinghydroxyl groups, hydroxyl groups on the hydrophilic particle can beactivated by replacing at least a portion of the hydroxyl groups with asulfonate group or chlorine. Exemplary sulfonate groups can be derivedfrom tresyl, mesyl, tosyl, or fosyl chloride, or any combinationthereof. Sulfonate can act to permit nucleophiles to replace thesulfonate. The sulfonate may further react with liberated chlorine toprovide chlorinated groups that can be used in a process to conjugatethe particles. In another example, amine groups on the hydrophilicpolymer can be activated.

For example, target analyte or analyte receptors can bind to thehydrophilic polymer through nucleophilic substitution with the sulfonategroup. In particular example, target analyte receptors terminated with anucleophile, such as an amine or a thiol, can undergo nucleophilicsubstitution to replace the sulfonate groups on the surface of thehydrophilic polymer. As a result of the activation, a conjugatedparticle can be formed.

In another example, where the particles are formed from monomerscomprising an amine, nucleophilic amino groups can be modified withdi-functional bis-electrophilic moieties, such as a di-isocyanate orbis-NHS ester, resulting in a hydrophilic particle reactive tonucleophiles.

When conjugated to polynucleotides, the polymer particles may include adensity of polynucleotides, termed nucleotide density, of at least 7×10⁴per μm³. For example, the nucleotide density may be at least 10⁵ perμm³, such as at least 10⁶ per μm³, at least 5×10⁶ per μm³, at least8×10⁶ per μm³, at least 1×10⁷ per μm³, or even at least 3×10⁷ per μm³.In a further example, the nucleotide density may be not greater than10¹⁵ per μm³.

Such polymer particles can be used in a variety of separationstechniques and analytic techniques. In particular, the polymer particlesmay be useful in binding polynucleotides. Such binding polynucleotidesmay be useful in separating polynucleotides from solution or can be usedfor analytic techniques, such as sequencing. In a particular exampleillustrated in FIG. 5, such polymeric particles can be used as a supportfor polynucleotides during sequencing techniques. For example, theparticles may immobilize a polynucleotide for sequencing usingfluorescent sequencing techniques. In another example, the polymerparticles may immobilize a plurality of copies of a polynucleotide forsequencing using ion-sensing techniques.

In general, the polymeric particle can be treated to include abiomolecule, including nucleosides, nucleotides, nucleic acids(oligonucleotides and polynucleotides), polypeptides, saccharides,polysaccharides, lipids, or derivatives or analogs thereof. For example,the polymer particles may bind or attach to a biomolecule. A terminalend or any internal portion of a biomolecule can bind or attach to apolymeric particle. The polymer particles may bind or attach to abiomolecule using linking chemistries. A linking chemistry includescovalent or non-covalent bonds, including an ionic bond, hydrogen bond,affinity bond, dipole-dipole bond, van der Waals bond, and hydrophobicbond. A linking chemistry includes affinity between binding partners,for example between: an avidin moiety and a biotin moiety; an antigenicepitope and an antibody or immunologically reactive fragment thereof; anantibody and a hapten; a digoxigen moiety and an anti-digoxigenantibody; a fluorescein moiety and an anti-fluorescein antibody; anoperator and a repressor; a nuclease and a nucleotide; a lectin and apolysaccharide; a steroid and a steroid-binding protein; an activecompound and an active compound receptor; a hormone and a hormonereceptor; an enzyme and a substrate; an immunoglobulin and protein A; oran oligonucleotide or polynucleotide and its corresponding complement.

As illustrated in FIG. 5, a plurality of polymer particles 204 (forexample monodisperse crosslinked hydrogel polymer particles of thedisclosure) may be placed in a solution along with a plurality ofpolynucleotides 202. The plurality of particles 204 may be activated orotherwise prepared to bind with the polynucleotides 202. For example,the particles 204 may include an oligonucleotide complementary to aportion of a polynucleotide of the plurality of polynucleotides 202.

In a particular embodiment, the polymer particles and polynucleotidesare subjected to polymerase chain reaction (PCR) amplification. Forexample, dispersed phase droplets 206 or 208 are formed as part of anemulsion and can include a particle or a polynucleotide. In an example,the polynucleotides 202 and the hydrophilic particles 204 are providedin low concentrations and ratios relative to each other such that asingle polynucleotide 202 is likely to reside within the same dispersedphase droplets as a single polymer particle 204. Other droplets, such asa droplet 208, can include a single polymer particle and nopolynucleotide. Each droplet 206 or 208 can include enzymes,nucleotides, salts or other components sufficient to facilitateduplication of the polynucleotide. Alternatively, amplificationtechniques, such as recombinase polymerase amplification (RPA) with orwithout emulsion, may be used.

In an aspect, the invention provides the use of crosslinked hydrogelpolymer particles of the invention in nucleic acid amplification.

In embodiments, methods for nucleic acid amplification comprise:conducting a primer extension reaction on a polynucleotide that ishybridized to an oligonucleotide which is attached to a polymerparticle. In embodiments the polymer particle is a monodispersecrosslinked hydrogel polymer particle of the disclosure. In embodiments,methods for nucleic acid amplification comprise: (a) providing a polymerparticle attached to a single-stranded oligonucleotide (e.g., a primeroligonucleotide); (b) providing a single-stranded templatepolynucleotide; (c) hybridising the single-stranded oligonucleotide tothe single-stranded template polynucleotide; (d) contacting thesingle-stranded template polynucleotide with a polymerase and at leastone nucleotide under conditions suitable for the polymerase to catalysepolymerisation of at least one nucleotide onto the single-strandedoligonucleotide so as to generate an extended single-strandedoligonucleotide. In embodiments, the method further comprises: (e)removing (e.g., denaturing) the single-stranded template polynucleotidefrom the extended single-stranded oligonucleotide so that thesingle-stranded oligonucleotide remains attached to the polymericparticle; (f) hybridising the remaining single-stranded oligonucleotideto a second single-stranded template polynucleotide; and (g) contactingthe second single-stranded template polynucleotide with a secondpolymerase and a second at least one nucleotide, under conditionssuitable for the second polymerase to catalyse polymerisation of thesecond at least one nucleotide onto the single-stranded oligonucleotideso as to generate a subsequent extended single-stranded oligonucleotide.In embodiments, steps (e), (f) and (g) can be repeated at least once. Inembodiments, the polymerase and the second polymerase comprise athermostable polymerase. In embodiments, the conditions suitable fornucleotide polymerisation include conducting the nucleotidepolymerisation steps (e.g., steps (d) or (g)) at an elevatedtemperature. In embodiments, the conditions suitable for nucleotidepolymerisation include conducting the nucleotide polymerisation step(e.g., steps (d) or (g)) at alternating temperatures (e.g., an elevatedtemperature and a relatively lower temperature). In embodiments, thealternating temperature ranges from 60-95° C. In embodiments, thetemperature cycles can be about 10 seconds to about 5 minutes, or about10 minutes, or about 15 minutes, or longer. In embodiments, methods fornucleic acid amplification can generate one or more polymeric particleseach attached to a plurality of template polynucleotides comprisingsequences that are complementary to the single-stranded templatepolynucleotide or to the second single-stranded template polynucleotide.In embodiments, each of the polymeric particles can be attached with aplurality of single-stranded oligonucleotides (e.g., captureoligonucleotides). In embodiments, step (b), (c), (d), (e), (f) or (g)can be conducted with a plurality of single-stranded polynucleotides. Inembodiments, at least a portion of the single-stranded oligonucleotidecomprises a nucleotide sequence that is complementary (or partiallycomplementary) to at least a portion of the single-strandedpolynucleotide. In embodiments, methods for nucleic acid amplification(as described above) can be conducted in an aqueous phase solution in anoil phase (e.g., dispersed phase droplet).

Following PCR, particles are formed, such as particle 210, which caninclude the polymer particle 212 and a plurality of copies 214 of thepolynucleotide. The majority of the polynucleotides 214 are illustratedon the external surface of the particle 210 for the purposes of clarity.The polynucleotides may, however, extend within (or be located within)the particle 210, as also illustrated. For example, hydrogel andhydrophilic particles may have a low concentration of polymer relativeto water and may therefore be relatively porous. They may includepolynucleotide segments on the interior of and throughout the particle210 and polynucleotides may reside in pores and other openings. Inparticular, the particle 210 can permit diffusion of enzymes,nucleotides, primers and reaction products used to monitor the reaction.A high number of polynucleotides per particle produces a better signal.

In embodiments, polymeric particles from an emulsion-breaking proceduremay be collected and washed in preparation for sequencing. Collectionmay be conducted by contacting biotin moieties (e.g., linked toamplified polynucleotide templates which are attached to the polymericparticles) with avidin moieties, and separation away from polymericparticles lacking biotinylated templates. Collected polymer particlesthat carry double-stranded template polynucleotides may be denatured toyield single-stranded template polynucleotides for sequencing.Denaturation steps may include treatment with base (e.g., NaOH),formamide, or pyrrolidone.

Sequencing can be performed by detecting nucleotide addition. Nucleotideaddition may be detected using methods such as fluorescent emissionmethods or ion detection methods. For example, a set of fluorescentlylabeled nucleotides may be provided to the system 216 and can migrate tothe well 218. Excitation energy may also be provided to the well 218.When a nucleotide is captured by a polymerase and added to the end of anextending primer, a label of the nucleotide may fluoresce, indicatingwhich type of nucleotide is added. These and other sequencing methodsdescribed herein may be combined with methods for nucleic acidamplification. For example, in the methods for nucleic acidamplification described herein with steps (a)-(g), the methods maycomprise a step (h) of sequencing by detecting the nucleotide addition.

In an alternative example, solutions including a single type ofnucleotide can be fed sequentially. In response to nucleotide addition,the pH within the local environment of the well 218 may change. Such achange in pH may be detected by ion sensitive field effect transistors(ISFET). As such, a change in pH can be used to generate a signalindicating the order of nucleotides complementary to the polynucleotideof the particle 210.

In particular, a sequencing system can include a well, or a plurality ofwells, disposed over a sensor pad of an ionic sensor, such as a fieldeffect transistor (FET). In embodiments, a system includes one or morepolymer particles loaded into a well which is disposed over a sensor padof an ionic sensor (e.g., FET), or one or more polymeric particlesloaded into a plurality of wells which are disposed over sensor pads ofionic sensors (e.g., FET). In embodiments, a FET may be a chemFET or anISFET. A “chemFET” or chemical field-effect transistor, includes a typeof field effect transistor that acts as a chemical sensor. The chemFEThas the structural analog of a MOSFET transistor, where the charge onthe gate electrode is applied by a chemical process. An “ISFET” orion-sensitive field-effect transistor, can be used for measuring ionconcentrations in solution; when the ion concentration (such as H⁺)changes, the current through the transistor changes accordingly.

Returning to FIG. 5, in an example, a well 218 of the array of wells maybe operatively connected to measuring devices. For example, forfluorescent emission methods, a well 218 may be operatively coupled to alight detection device. In the case of ionic detection, the lowersurface of the well 218 may be disposed over a sensor pad of an ionicsensor, such as a field effect transistor (e.g. a chemFET).

Exemplary systems involving sequencing via detection of ionic byproductsof nucleotide incorporation are the Ion Torrent PGM™ or Proton™sequencers (Life Technologies), which are ion-based sequencing systemsthat sequences nucleic acid templates by detecting hydrogen ionsproduced as a byproduct of nucleotide incorporation. Typically, hydrogenions are released as byproducts of nucleotide incorporations occurringduring template-dependent nucleic acid synthesis by a polymerase. TheIon Torrent PGM™ or Proton™ sequencers detect the nucleotideincorporations by detecting the hydrogen ion byproducts of thenucleotide incorporations. The Ion Torrent PGM™ or Proton™ sequencersmay include a plurality of template polynucleotides to be sequenced,each template disposed within a respective sequencing reaction well inan array. The wells of the array may each be coupled to at least one ionsensor that can detect the release of H⁺ ions or changes in solution pHproduced as a byproduct of nucleotide incorporation. The ion sensorcomprises a field effect transistor (FET) coupled to an ion-sensitivedetection layer that can sense the presence of H⁺ ions or changes insolution pH. The ion sensor may provide output signals indicative ofnucleotide incorporation which can be represented as voltage changeswhose magnitude correlates with the H⁺ ion concentration in a respectivewell or reaction chamber. Different nucleotide types may be flowedserially into the reaction chamber, and may be incorporated by thepolymerase into an extending primer (or polymerization site) in an orderdetermined by the sequence of the template. Each nucleotideincorporation may be accompanied by the release of H⁺ ions in thereaction well, along with a concomitant change in the localized pH. Therelease of H⁺ ions may be registered by the FET of the sensor, whichproduces signals indicating the occurrence of the nucleotideincorporation. Nucleotides that are not incorporated during a particularnucleotide flow may not produce signals. The amplitude of the signalsfrom the FET can also be correlated with the number of nucleotides of aparticular type incorporated into the extending nucleic acid moleculethereby permitting homopolymer regions to be resolved. Thus, during arun of the sequencer, multiple nucleotide flows into the reactionchamber along with incorporation monitoring across a multiplicity ofwells or reaction chambers may permit the instrument to resolve thesequence of many nucleic acid templates simultaneously.

Another sequencing system is the SOLiD™ sequencing system (Sequencing byOligonucleotide Ligation and Detection) of Applied Biosystems, whichuses stepwise cycled ligation for high throughput DNA sequencing. Inthis bead based system, beads (i.e. polymer particles) loaded with DNAtemplates undergo sequential ligation and cleavage reactions using4-colour, fluorescently-labeled octameric probes. These probes aredelivered serially and serve to interrogate dinucleotide positions onDNA strands. It would be desirable to support higher bead densities thatfacilitate an increased number of bead events per instrument run andimproved probe chemistry, affording increased sequencing fidelity.

Sequencing by Oligonucleotide Ligation and Detection involves attachmentof a nucleic acid target to a crosslinked polymer particles (beads)followed by immobilization of a plurality of the particles onto asurface. Each nucleic acid-bead conjugate comprises a unique DNAsequence, sequencing techniques of this type are disclosed inInternational Publication No. WO 2006/084132 A2 (incorporated herein byreference).

Methods of attachment of the beads to the support have utilized a flatglass microscope slide irreversibly coated with streptavidin. Nucleicacid-laden beads are contacted with biotinylated nucleotides (e.g.,obtained by the action of biotinylated dNTP's and terminaldeoxytransferase on the DNA target subsequent to attachment to thebead). Incubation of the biotinylated beads with the streptavidin coatedslide results in immobilization of the beads onto the slide by theinteraction of streptavidin with the biotin. While kinetically this is avery effective attachment scheme, movement of the beads on the slide wassometimes observed under the conditions required by the DNA sequenceassay. When beads are present in high densities on the slide (e.g., upto 100,000 beads/mm²) and interrogated multiple times (e.g., up to 25times), any significant bead movement can preclude robust identificationof a particular bead on subsequent scans within a dense population ofbeads.

US 2009/0099027 (equivalent to WO2009/026546, both incorporated hereinby reference) therefore describes a covalent system for beadimmobilization that reduces movement of the beads during sequencing andother forms of genetic analysis. The method comprises: reacting anucleophilic group on the surface of a substrate with a moleculecomprising a plurality of electrophilic groups thereby providing one ormore free electrophilic groups on the surface of the substrate; andreacting nucleophilic groups on a surface of a particulate material withthe one or more free electrophilic groups on the surface of thesubstrate to covalently attach the particulate material to thesubstrate.

US 2009/0099027 describes the modification of a nucleophilic (moreparticularly, amino functional) surface with a multifunctionalelectrophilic reagent. For example, the electrophilic surfaces ofsilicate glass microscope slides can be readily converted to anucleophilic surface by reacting surface groups with (aminopropyl)trialkoxysilanes.

A DNA target nucleic acid that had been covalently attached to acrosslinked polymer bead may be modified by the action of aminoalkyldNTP's and terminal deoxytransferase on the DNA target subsequent toattachment to the bead. The nucleophilic amino group on the DNA targetcan then react with the residual electrophilic group of the supportsurface to form multiple stable covalent bonds between the bead and theglass surface.

It has been found that stable covalent bonds can be formed between asurface containing electrophilic groups and particles containingnucleophilic groups. In addition, beads containing nucleophilic aminogroups from the action of amino-dNTP's and terminal deoxytransferase ona DNA target can be immobilized under aqueous basic conditions on themodified surface. For example, surfaces comprising amino groups thathave been activated with benzene 1,4-diisothiocyanate can be used toimmobilize beads with nucleophilic groups. In addition, the covalentattachment appears to be quite stable, and no bead movement is observed.

The surface immobilized beads can be used in methods of analysingnucleic acid sequences based on repeated cycles of duplex extensionalong a single stranded template via ligation. Sequencing methods ofthis type are disclosed in U.S. Pat. Nos. 5,750,341; 5,969,119; and6,306,597 B1 and in International Publication No. WO 2006/084132 A2.Each of these publications is incorporated by reference herein in itsentirety. Moreover, the techniques described in the aforementionedpublications can be used to analyse (e.g., sequence) nucleic acidtemplates attached to particles that are bound to supports as describedherein. The immobilized beads can be used in sequencing methods that donot necessarily employ a ligation step, such as sequencing using labelednucleotide that have removable blocking groups that preventpolynucleotide chain extension (e.g., U.S. Pat. Nos. 6,664,079;6,232,465; and 7,057,026, each of which is incorporated by referenceherein in its entirety). The immobilized beads can be used in a varietyof techniques in which signals on the beads are repeated detectedthrough multiple cycles.

The beads which are used in SOLiD sequencing may be monodispersecrosslinked hydrogel particles of the disclosure. An embodimenttherefore includes the use of the monodisperse particles in the methodsand products disclosed in the publications mentioned in the previousparagraph and the applicant of the present application considers allsuch uses, methods and products to fall within the present invention andreserves the right to claim them. The use of submicron particles inSOLiD sequencing enables a greater density of particles to be attachedto the glass surfaces (e.g. glass panels or microscope slides). Furtherincluded in an embodiment is a method of performing SOLiD sequencingwhich uses monodisperse particles of the disclosure, e.g. whereinmonodisperse submicron of the present disclosure are coupled to anucleic acid target and immobilised on a surface, e.g. a glass surface.The method of immobilisation is not critical and may be covalent ornon-covalent, examples of non-covalent coupling being throughstreptavidin/avidin-biotin binding. The covalent coupling may be asdescribed in US 2009/0099027 and WO2009026546, for example, but anyother suitable technique for covalent coupling may be used. Included inan embodiment, therefore, is a method of forming a product (an articleof manufacture), comprising coupling monodisperse submicron particles ofthe present disclosure to a nucleic acid and optionally furthercomprising immobilising the resultant nucleic acid-laden particles on asurface, e.g. a glass surface. The nucleic acid may be used as a targetin sequencing, e.g. using SOLiD sequencing.

For example, a method is provided that comprises:

(a) hybridizing a first initializing oligonucleotide probe to a targetpolynucleotide to form a probe-target duplex, wherein theoligonucleotide probe has an extendable probe terminus, wherein thetarget polynucleotide is attached to a polymer particle which is amember of a population of polymer particles as disclosed herein andwherein the particle is covalently attached to the surface of a solidsupport;

(b) ligating a first end of an extension oligonucleotide probe to theextendable probe terminus thereby forming an extended duplex containingan extended oligonucleotide probe, wherein the extension oligonucleotideprobe comprises a cleavage site and a detectable label;

(c) identifying one or more nucleotides in the target polynucleotide bydetecting the label attached to the just-ligated extensionoligonucleotide probe;

(d) cleaving the just-ligated extension oligonucleotide probe at thecleavage site to generate the extendable probe terminus, whereincleavage removes a portion of the just-ligated extension oligonucleotideprobe that comprises the label from the probe-target duplex; and

(e) repeating steps (b), (c) and (d) until a sequence of nucleotides inthe target polynucleotide is determined.

Also provided is a method of sequencing a nucleic acid comprising:

(a) hybridizing a primer to a target polynucleotide to form aprimer-target duplex, wherein the target polynucleotide is attached at a5′ end to a polymer particle which is a member of a population ofpolymer particles as disclosed herein and wherein the polymer particleis covalently attached to the surface of a support;(b) contacting the primer-target duplex with a polymerase and one ormore different nucleotide analogues to incorporate a nucleotide analogueonto the 3′ end of the primer thereby forming an extended primer strand,wherein the incorporated nucleotide analogue terminates the polymerasereaction and wherein each of the one or more nucleotide analoguescomprises (i) a base selected from the group consisting of adenine,guanine, cytosine, thymine and uracil and their analogues (ii) a uniquelabel attached to the base or analogue thereof via a cleavable linker;(iii) a deoxyribose; and (iv) a cleavable chemical group which caps an—OH group at a 3′-position of the deoxyribose;(c) washing the surface of the support to remove any unincorporatednucleotide analogues;(d) detecting the unique label attached to the just-incorporatednucleotide analogue to thereby identify the just-incorporated nucleotideanalogue;(e) optionally, permanently capping any unreacted —OH group on theextended primer strand;(f) cleaving the cleavable linker between the just incorporatednucleotide analogue and the unique label;(g) cleaving the chemical group capping the —OH group at the 3′-positionof the deoxyribose of the just incorporated nucleotide analogue to uncapthe —OH group;(h) washing the surface of the support to remove cleaved compounds;(i) repeating steps (b)-(h).

The polymer particles of the disclosure may be used in any method ofnucleic acid sequencing which involves a polymer particle. An embodimentincludes particles of the disclosure coupled to a nucleic acid as wellas a method of sequencing a nucleic acid which comprises coupling anucleic acid to a population of particles of the disclosure. The nucleicacid may be DNA or RNA.

The present disclosure includes a product (e.g. an article ofmanufacture) comprising a plurality of monodisperse particles of thedisclosure coupled to a substrate such as, for example, glass surface,for example through a streptavidin-biotin linkage, an avidin-biotinlinkage or through a covalent linkage, e.g. as described in US2009/0099027 and WO2009/026546. The particles may be coupled to thesubstrate through a nucleic acid. The present disclosure includes theuse of the monodisperse submicron particles of the disclosure to makesuch a product. An embodiment includes the use of the attachmentchemistry described in US 2009/0099027 and WO2009/026546 to attachmonodisperse submicron particles of the disclosure to a substrate, andthe applicant reserves the right to claim methods of using suchchemistry and the products thereof. The present specification thereforeincludes by reference the disclosures of US 2009/0099027 andWO2009026546.

Embodiments therefore include methods in which functionalisedmonodisperse polymer particles of the disclosure are subjected to one ormore further reactions to obtain a desired product. Other embodimentsinclude the use of these products in applications.

In view of the consistency of the quality and characteristics which theparticles of the disclosure may possess, they may be used in methodswhich comprise performing processes in relation to a conjugatedsubstance, e.g. selected from labels, biological molecules andbiological structures, for example biological molecules such as aminoacids, saccharides, nucleotides and nucleosides and multimers made bycondensing together two or more such monomers, e.g. polypeptides,proteins, polysaccharides, oligonucleotides and nucleic acids. As labelsmay be mentioned dyes, e.g. fluorescent dyes, quenchers, enzymes, andsemiconductor nanocrystals. Embodiments of the invention include suchuses as well as:

-   i) conjugates comprising a population of particles of the disclosure    at least a portion of which are coupled to a conjugated substance,    e.g. one as just described-   ii) a method comprising coupling at least a portion of a population    of particles of the disclosure to a substance, e.g. one as just    described-   iii) a method comprising coupling at least a portion of a population    of particles of the disclosure to a substrate.

Analytical Methods Molecular Weight Measurement

The weight average molecular weight (Mw) of the oligomers in the seedparticle can be determined from measurements made using gel permeationchromatography (GPC). In GPC a series of polymer particle standards arerun and used to generate a calibration curve. The Mw of the oligomersmay be measured by GPC relative to polystyrene standards using as eluentDMF with 0.01 M LiBr. As these Mw values are calculated relative tostandards of a polymer (polystyrene) that is different to that of theseed particles, the calculated Mw represents a relative value, ratherthan an absolute value. The measurements will therefore be reproducible,but will not provide the actual Mw.

An outline of the GPC method that was used in the examples providedherein is as follows. The following experimental conditions were used:

Eluent: DMF with 0.01 M LiBrPrecolumn: PSS GRAM, 10 μm, Guard ID 8.0 mm×50 mmColumns: PSS GRAM, 10 μm, Linear M ID 8.0 mm×300 mm PSS GRAM, 10 μm,Linear M ID 8.0 mm×300 mm

Temperature: 70° C.

Pump: PSS SECcurity 1260 HPLC pumpFlow rate: 1.0 mL/minInject. system: PSS SECcurity 1260 autosamplerInject. volume: 50 μLSample conc.: 3.0 g/LDetector: PSS SECcurity 1260 refractive index detector (RID)Chromatography data system: PSS WinGPC UniChrom Version 8.2Polystyrene standards with different molecular weights were measuredunder the above experimental conditions to obtain a calibration curve.The samples were then run. Mw was then calculated for the samples basedon the PS calibration curve.

Size and Size Distribution

The size distribution of samples can be measured using disccentrifugation, e.g. CPS Disc Centrifugation™ on Disc Centrifuge ModelDC20000, using protocols provided by the instrument manufacturer.Accurate results require calibration with a standard of similar densityto the sample being analysed and thus is only of use where a suitablepolymeric standard is available, for example a set of compactpolystyrene particle standards for particles of the disclosurecomprising predominantly polystyrene. Where the samples being measuredhave a density that is not known, e.g. for porous particles, themeasurement obtained by CPS disc centrifugation will be reproducible butwill not provide the actual diameter.

An outline of the CPS Disc Centrifugation™ that was used in the examplesprovided herein is as follows, with three different experimental methodsused, with selection of the appropriate method based on, e.g. the sizeand porosity of the particles to be analysed. The skilled person wouldbe able to readily adapt these methods as appropriate, for example byselecting a suitable gradient, disc speed and standard particles, basedon the size and porosity of the particles to be analysed.

CPS method 1 (e.g. Examples 32, 33): Disc centrifuge analysis wasperformed on a CPS DC20000 from CPS instruments with a disc speed of7500 rpm and a gradient of 3-7 wt % sucrose in 1.5 g/L SDS (aq.). Thegradient was made using an Auto Gradient pump from CPS instruments andthe volume of the injected gradient was 16-17 mL. The samples werediluted to approximately 0.01 wt % in MilliQ-H₂O prior to injection.The method used for analysis had the following settings: Max. diameter4.0 μm, min. diameter 0.2 μm, particles density 1.032 g/mL, particlerefractive index 1.4, particle absorption 0, particle non-sphericity 1,calibration standard diameter 1.069 μm, calibration standard density1.052, standard half-with 0.15 μm, liquid density 1.016 g/mL, liquidrefractive index 1.343.The size reported is the absorption peak diameter and the CV isdetermined by setting the boarders around the main peak.CPS method 2 (e.g. Examples 8, 9, 16-30): Disc centrifuge analysis wasperformed on a CPS DC20000 from CPS instruments with a disc speed of7500 rpm and a gradient of 3-7 wt % sucrose in 1.5 g/L SDS (aq.). Thegradient was made using an Auto Gradient pump from CPS instruments andthe volume of the injected gradient was 16-17 mL.The method used for analysis had the following settings: Max. diameter6.0 μm, min. diameter 0.05 μm, particles density 1.032 g/mL, particlerefractive index 1.032, particle absorption 0, particle non-sphericity1, calibration standard diameter 0.486 μm, calibration standard density1.052, standard half-with 0.15 μm, liquid density 1.016 g/mL, liquidrefractive index 1.343.The size reported is the absorption peak diameter and the CV isdetermined by setting the borders around the main peak.CPS method 3 (e.g. Examples 34-47): Disc centrifuge analysis wasperformed on a CPS DC20000 from CPS instruments with a disc speed of10000 rpm and a gradient of 8-24 wt % sucrose in 1.5 g/L SDS (aq.). Thegradient was made using an Auto Gradient pump from CPS instruments andthe volume of the injected gradient was 16-17 mL. The samples werediluted to approximately 0.01 wt % in MilliQ-H2O prior to injection.The method used for analysis had the following settings: Max. diameter4.0 μm, min. diameter 0.1 μm, particles density 1.6 g/mL, particlerefractive index 1.592, particle absorption 0.2, particle non-sphericity1, calibration standard diameter 1.098, calibration standard density1.6, standard half-with 0.2 μm, liquid density 1.06 g/mL, liquidrefractive index 1.355.The size reported is the absorption peak diameter and the CV isdetermined by setting the boarders around the main peak.

Photon correlation spectroscopy (PCS) can be used to obtain thehydrodynamic diameter of a particle in the form of the z-average. Themeasurement is independent of the particle density and based on Brownianmotion of small particles. PCS measurements for nanosized particles canbe obtained, for example with a Malvern ZetaSizer Nano-ZS, ModelZEN3600. Further details and methods can be found in the MalvernZetasizer Nano series manual (incorporated herein by reference in itsentirety).

Another technique that can be used to determine the size and sizedistribution of particles is optical microscopy. A population of beadsmay be prepared by placing an aqueous solution comprising beads on amicroscope slide, then capturing an image of the beads at a suitablelevel of magnification, e.g. 100× or greater, and analyzing the size ofthe beads using image analysis software.

An outline of the optical microscopy method that was used in theexamples provided herein is as follows. Microscopy samples were preparedby sandwiching a 0.12 mm, 9 mm diameter Secure-Sea™ spacer between acover slip and a Secure-Sea™ Hybridization Chamber Gasket. The formedcavity was filled with 8 μL of beads diluted in 150 mM NaCl followed bya short centrifugation and attachment to a microscope slide. Phasecontrast optical microscopy was then performed on an Olympus IX81inverted microscope using 100× lens. Multiple images were collectedautomatically using a Matlab based macro for μManager. An out of focusimage was collected as the background and subtracted from the phasecontrast image. The background subtracted image was then thresholdedusing Otsu's method to identify individual beads for size analysis.Green fluorescent polystyrene bead with standard sizes were used tocalibrate the measured bead size by fluorescence microscopy. Then aseries of Alexa 488 labeled hydrogel beads were used to calibrate phasecontrast size to fluorescent size.

Optical microscopy is a preferred method for measuring the size of theparticles, as it provides a measurement that is independent of thedensity of the particles. Disc centrifuge analysis is a preferred methodof measuring the size distribution of particles (CV), as opticalmicroscopy is based on image analysis and the presence of artefacts inthe image can result in an artificially high CV.

EXAMPLES Synthesis of Seed Particles Example 1

A 250 mL 3-necked round-bottom flask equipped with a mechanical bladestirrer and a condenser was charged with 5.0 g of distilledN,N-dimethylacrylamide, 4.0 g of Kraton G1650, 0.20 g of2,2′-Azobis(2-methylpropionitrile), 108.0 g of heptane, 27.1 g oftoluene, and 0.21 mL of 1-octanethiol. Once everything had dissolved thereaction mixture was purged with Ar (g) for 10 min. The reaction wasthen heated to 70° C. using an oil bath and heated for 8 h whilestirring at 400 rpm. The resulting dispersion was analyzed by dynamiclight scattering yielding a z-average diameter of 521 nm.

Example 2

A 500 mL 3-necked round-bottom flask equipped with a mechanical and ablade stirrer and a condensor was charged with 10.1 g of distilledN,N-dimethylacrylamide, 8.0 g of Kraton G1650, 0.40 g of2,2′-Azobis(2-methylpropionitrile), 216.0 g of heptane, 54.1 g oftoluene, and 0.85 mL of 1-octanethiol. Once everything had dissolved thereaction mixture was purged with Ar (g) for 10 min. The reaction wasthen heated to 70° C. using an oil bath and heated for 8 h whilestirring at 360 rpm. The resulting dispersion was analyzed by dynamiclight scattering yielding a z-average diameter of 733 nm. The particlespresent in the dispersion were also analysed by GPC yielding an Mw of8,410.

Example 3

A 2 L jacketed round-bottom flask equipped with a mechanical anchorstirrer and a condensor was charged with 36.6 g of distilledN,N-dimethylacrylamide, 27.7 g of Kraton G1650, 1.4 g of2,2′-Azobis(2-methylpropionitrile), 747.2 g of heptane, 186.9 g oftoluene, and 2.56 g of 1-octanethiol. Once everything had dissolved thereaction mixture was purged with Ar (g) for 30 min. The reaction wasthen heated to 71° C. using a temperature controlled water bathconnected to the reactor and heated for 15 h while stirring at 350 rpm.During the first 2.5 h of heating a low flow of Ar (g) was maintained.The resulting dispersion was analyzed by dynamic light scatteringyielding a z-average diameter of 1035 nm. The particles present in thedispersion were also analysed by GPC yielding an Mw of 10,300.

Example 4

The method was performed as per Example 1, but with the reaction mixtureheated and stirred at 350 rpm overnight. The resulting dispersion wasanalyzed by dynamic light scattering yielding a z-average diameter of387 nm. The particles present in the dispersion were also analysed byGPC yielding an Mw of 22,400.

Example 5 (Comparative Example)

The method was performed as per Example 4, but without the addition of1-octantethiol, i.e. the reaction mixture did not contain any1-octantethiol. The resulting dispersion was analyzed by dynamic lightscattering yielding a z-average diameter of 238 nm. The particlespresent in the dispersion were also analysed by GPC yielding an Mw of403,000.

Example 6

The method was performed as per Example 4. The resulting dispersion wasanalyzed by dynamic light scattering yielding a z-average diameter of358 nm. The particles present in the dispersion were also analysed byGPC yielding an Mw of 21,200.

Synthesis of Polymer Particles from Low Molecular Weight Seed Particles

Example 7

100 mL batch of micron-sized acrylamide particles formed inHeptane/Toluene using 0.8 wt % initiator.

Solution 1 (10 wt % Acrylamide (AAm) stock solution): 50 g acrylamidemonomer and 450 g water are mixed until a clear and homogeneous solutionis formed.

Solution 2 (stabilizer): 10 g of Hypermer 2296 (stabilizer) is weighedout followed by the addition of 70 g of a 1:1 (vol:vol) heptane:toluene(hereafter referred to as the solvent or H/T) mixture. The stabilizersolution is stirred until a homogeneous solution is obtained. 0.8 g ofAzobisdimethyl valeronitril (radical initiator from Wako Chemicals,hereafter called V-65) is added to the solution under gentle stirring.

Solution 3 (monomer phase): 0.5 g of 1,2-dihydroxy bis acrylamide(crosslinker, hereafter called DHEBA) is added to 20 g of solution 1 (10wt % AAm stock solution). The solution is then heated to −40° C. tofacilitate mixing.

Solution 4 (emulsion phase): Solutions 2 and 3 are mixed usingUltraturrax for 1 minute, and then sonicated for 3 minutes (6×30 secintervals) on ice (Branson digital sonifier 450 CE, 40% amplitude).Following emulsification, Solution 4 is transferred to a 100 mL reactorwith stirrer, and 8.1 g of a suspension of seed particles of Example 2(approximately 7 wt % dry content—hereafter referred to as seed) isadded to the emulsion, and the seed-containing emulsion is allowed toswell overnight (˜16 h) at room temperature under gentlestirring/rotation). Following swelling, Solution 4 is polymerized at 50°C. for 7 h under stirring.

Example 8

10 mL batch of micron-sized acrylamide particles formed inHeptane/Toluene using 0.5 wt % initiator.

Solution 1 (10 wt % Acrylamide (AAm) stock solution): 50 g acrylamidemonomer and 450 g water are mixed until a clear and homogeneous solutionis formed.

Solution 2 (stabilizer): 1 g of Hypermer 2296 (stabilizer) is weighedout followed by the addition of 7 g of a 1:1 (vol:vol) heptane:toluene(hereafter referred to as the solvent or H/T) mixture. The stabilizersolution is stirred until a homogeneous solution is obtained. 0.05 g ofAzobisdimethyl valeronitril (radical initiator from Wako Chemicals,hereafter called V-65) is added to the solution under gentle stirring.

Solution 3 (monomer phase): 0.05 g of 1,2-dihydroxy bis acrylamide(crosslinker, hereafter called DHEBA) is added to 2 g of solution 1 (10wt % AAm stock solution). The solution is then heated to ˜40° C. tofacilitate mixing.

Solution 4 (emulsion phase): Solutions 2 and 3 are mixed usingUltraturrax for 1 minute, and then sonicated for 3 minutes (6×30 secintervals) on ice (Branson digital sonifier 450 CE, 40% amplitude).Following emulsification, 0.81 g of a suspension of seed particles ofExample 2 (approximately 7 wt % dry content—hereafter referred to asseed) is added to the emulsion, and the seed-containing emulsion isallowed to swell overnight (˜16 h) at room temperature under gentlestirring/rotation). Following swelling, Solution 4 is polymerized at 50°C. for 7 h under stirring.

Redispersion and analysis: To a 2 mL eppendorf tube 300 μL ofpolymerized emulsion, 300 μL of a heptane/toluene mixture (50:50), and1200 μL of a methanol/deionized water mixture (50:50) were added. Thetube was vortexed and centrifuged. The supernatant was removed and thepellet was washed three times with 300 μL heptane/toluene mixture(50:50) and 1200 μL of a methanol/deionized water mixture (50:50).Subsequently one wash in a methanol/deionized water mixture (50:50) andtwo washes in deionized water were performed before finally resuspendingthe pellet in 200 μL of deionized water. The product was analyzed usingoptical microscopy and disc centrifuge. Microscopy was performed bydiluting the redispersed product 20 times in 150 mM NaCl solution andimage statistics was collected from 28 images yielding a mode diameterof 2.3 μm. Relative disc centrifuge diameter using CPS method 2 wasdetermined to 2.8 μm with a CV=3.0% (main peak).

Example 9

10 mL batch of micron-sized acrylamide particles formed inHeptane/Toluene using alternative stabilizer.

Solution 1 (10 wt % Acrylamide (AAm) stock solution): 50 g acrylamidemonomer and 450 g water are mixed until a clear and homogeneous solutionis formed.

Solution 2 (stabilizer): 1 g of Abil EM 90 (stabilizer) is weighed outfollowed by the addition of 7 g of a 1:1 (vol:vol) heptane:toluene(hereafter referred to as the solvent or H/T) mixture. The stabilizersolution is stirred until a homogeneous solution is obtained. 0.08 g ofAzobisdimethyl valeronitril (radical initiator from Wako Chemicals,hereafter called V-65) is added to the solution under gentle stirring.

Solution 3 (monomer phase): 0.05 g of 1,2-dihydroxy bis acrylamide(crosslinker, hereafter called DHEBA) is added to 2 g of solution 1 (10wt % AAm stock solution). The solution is then heated to −40° C. tofacilitate mixing.

Solution 4 (emulsion phase): Solutions 2 and 3 are mixed usingUltraturrax for 1 minute, and then sonicated for 3 minutes (6×30 secintervals) on ice (Branson digital sonifier 450 CE, 40% amplitude).Following emulsification, 0.81 g of a suspension of seed particles ofExample 2 (approximately 7 wt % dry content—hereafter referred to asseed) is added to the emulsion, and the seed-containing emulsion isallowed to swell overnight (˜16 h) at room temperature under gentlestirring/rotation). Following swelling, Solution 4 is polymerized at 50°C. for 7 h under stirring.

Redispersion and analysis: To a 2 mL eppendorf tube 300 μL ofpolymerized emulsion was added, the tube filled with toluene to the 2 mLmark and centrifuged. After removal of the supernatant the pellet wasredispersed in toluene and centrifuged. Additionally two washes usingtoluene was performed followed by two washes using deionized water.Finally the pellet was redispersed in deionized water to a total volumeof 2 mL and the product was analyzed using optical microscopy and disccentrifuge. Microscopy image statistics was collected from 10 imagesyielding a mode diameter of 2.6 μm. Relative disc centrifuge diameterusing CPS method 2 was determined to 3.3 μm with a CV=3.9%.

Example 10

10 mL batch of micron-sized acrylamide particles formed in nonpolarsolvent using with swelling at elevated temperature.

Solution 1 (10 wt % Acrylamide (AAm) stock solution): 50 g acrylamidemonomer and 450 g water are mixed until a clear and homogeneous solutionis formed.

Solution 2 (stabilizer): 1 g of Hypermer 2296 (stabilizer) is weighedout followed by the addition of 7 g of a 1:1 (vol:vol) heptane:toluene(hereafter referred to as the solvent or H/T) mixture. The stabilizersolution is stirred until a homogeneous solution is obtained.

Solution 3 (monomer phase): 0.05 g of 1,2-dihydroxy bis acrylamide(crosslinker, hereafter called DHEBA) is added to 2 g of solution 1 (10wt % AAm stock solution). The solution is then heated to −40° C. tofacilitate mixing.

Solution 4 (emulsion phase): Solutions 2 and 3 are mixed usingUltraturrax for 1 minute, and then sonicated for 3 minutes (6×30 secintervals) on ice (Branson digital sonifier 450 CE, 40% amplitude).Following emulsification, 0.81 g of a suspension of seed particles ofExample 2 (approximately 7 wt % dry content—hereafter referred to asseed) is added to the emulsion, and the seed-containing emulsion isallowed to swell overnight (˜16 h) at 50° C. under gentlestirring/rotation). Following swelling, 0.08 g of Azobisdimethylvaleronitril (radical initiator from Wako Chemicals, hereafter calledV-65) is added to the solution under gentle stirring. Solution 4 ispolymerized at 50° C. for 7 h under stirring.

Example 11

10 mL batch of micron-sized acrylamide particles formed inHeptane/Toluene using higher acrylamide content.

Solution 1 (40 wt % Acrylamide (AAm) stock solution): 200 g acrylamidemonomer and 300 g water are mixed until a clear and homogeneous solutionis formed.

Solution 2 (stabilizer): 1 g of Hypermer 2296 (stabilizer) is weighedout followed by the addition of 7 g of a 1:1 (vol:vol) heptane:toluene(hereafter referred to as the solvent or H/T) mixture. The stabilizersolution is stirred until a homogeneous solution is obtained.

Solution 3 (monomer phase): 0.05 g of 1,2-dihydroxy bis acrylamide(crosslinker, hereafter called DHEBA) is added to 2 g of solution 1 (10wt % AAm stock solution). The solution is then heated to ˜40° C. tofacilitate mixing.

Solution 4 (emulsion phase): Solutions 2 and 3 are mixed usingUltraturrax for 1 minute, and then sonicated for 3 minutes (6×30 secintervals) on ice (Branson digital sonifier 450 CE, 40% amplitude).Following emulsification, 0.81 g of a suspension of seed particles ofExample 2 (approximately 7 wt % dry content—hereafter referred to asseed) is added to the emulsion, and the seed-containing emulsion isallowed to swell overnight (˜16 h) at 50° C. under gentlestirring/rotation). Following swelling, 0.08 g of Azobisdimethylvaleronitril (radical initiator from Wako Chemicals, hereafter calledV-65) is added to the solution under gentle stirring. Solution 4 ispolymerized at 50° C. for 7 h under stirring.

Example 12

10 mL batch of micron-sized acrylamide particles formed in Linpar 10-13.

Solution 1 (10 wt % Acrylamide (AAm) stock solution): 50 g acrylamidemonomer and 450 g water are mixed until a clear and homogeneous solutionis formed.

Solution 2 (stabilizer): 1 g of Hypermer 2296 (stabilizer) is weighedout followed by the addition of 7 g Linpar 10-13 (hereafter referred toas the solvent). The stabilizer solution is stirred until a homogeneoussolution is obtained.

Solution 3 (monomer phase): 0.05 g of 1,2-dihydroxy bis acrylamide(crosslinker, hereafter called DHEBA) is added to 2 g of solution 1 (10wt % AAm stock solution). The solution is then heated to ˜40° C. tofacilitate mixing.

Solution 4 (emulsion phase): Solutions 2 and 3 are mixed usingUltraturrax for 1 minute, and then sonicated for 3 minutes (6×30 secintervals) on ice (Branson digital sonifier 450 CE, 40% amplitude).Following emulsification, 0.81 g of a PDMAAm particle suspension(approximately 7 wt % dry content—hereafter referred to as seed) isadded to the emulsion, and the seed-containing emulsion is allowed toswell overnight (˜16 h) at 50° C. under gentle stirring/rotation).Following swelling, 0.08 g of Azobisdimethyl valeronitril (radicalinitiator from Wako Chemicals, hereafter called V-65) is added to thesolution under gentle stirring. Solution 4 is polymerized at 50° C. for7 h under stirring.

Example 13

10 mL batch of micron-sized hydroxymethyl acrylamide particles formed inHeptane/Toluene.

Solution 1 (10 wt % N-hydroxymethylacrylamide (HMAAm) stock solution):50 g hydroxymethyl acrylamide monomer and 450 g water are mixed until aclear and homogeneous solution is formed.

Solution 2 (stabilizer): 1 g of Hypermer 2296 (stabilizer) is weighedout followed by the addition of 7 g of a 1:1 (vol:vol) heptane:toluene(hereafter referred to as the solvent or H/T) mixture. The stabilizersolution is stirred until a homogeneous solution is obtained.

Solution 3 (monomer phase): 0.05 g of 1,2-dihydroxy bis acrylamide(crosslinker, hereafter called DHEBA) is added to 2 g of solution 1 (10wt % HMAAm stock solution). The solution is then heated to ˜40° C. tofacilitate mixing. 0.08 g of Azobisdimethyl valeronitril (radicalinitiator from Wako Chemicals, hereafter called V-65) is added to thesolution under gentle stirring.

Solution 4 (emulsion phase): Solutions 2 and 3 are mixed usingUltraturrax for 1 minute, and then sonicated for 3 minutes (6×30 secintervals) on ice (Branson digital sonifier 450 CE, 40% amplitude).Following emulsification, 0.81 g of a suspension of seed particles ofExample 2 (approximately 7 wt % dry content—hereafter referred to asseed) is added to the emulsion, and the seed-containing emulsion isallowed to swell overnight (˜16 h) at room temperature under gentlestirring/rotation). Following swelling, Solution 4 is polymerized at 50°C. for 7 h under stirring.

Example 14

10 mL batch of micron-sized TRIS acrylamide particles formed inHeptane/Toluene.

Solution 1 (10 wt % TRIS acrylamide (TRIS) stock solution): 50 g TRISacrylamide monomer and 450 g water are mixed until a clear andhomogeneous solution is formed.

Solution 2 (stabilizer): 1 g of Hypermer 2296 (stabilizer) is weighedout followed by the addition of 7 g of a 1:1 (vol:vol) heptane:toluene(hereafter referred to as the solvent or H/T) mixture. The stabilizersolution is stirred until a homogeneous solution is obtained. 0.08 g ofAzobisdimethyl valeronitril (radical initiator from Wako Chemicals,hereafter called V-65) is added to the solution under gentle stirring.

Solution 3 (monomer phase): 0.05 g of 1,2-dihydroxy bis acrylamide(crosslinker, hereafter called DHEBA) is added to 2 g of solution 1 (10wt % TRIS stock solution). The solution is then heated to ˜40° C. tofacilitate mixing.

Solution 4 (emulsion phase): Solutions 2 and 3 are mixed usingUltraturrax for 1 minute, and then sonicated for 3 minutes (6×30 secintervals) on ice (Branson digital sonifier 450 CE, 40% amplitude).Following emulsification, 0.81 g of a suspension of seed particles ofExample 2 (approximately 7 wt % dry content—hereafter referred to asseed) is added to the emulsion, and the seed-containing emulsion isallowed to swell overnight (˜16 h) at room temperature under gentlestirring/rotation). Following swelling, Solution 4 is polymerized at 50°C. for 7 h under stirring.

Example 15

10 mL batch of micron-sized Hydroxyethyl methacrylate particles formedin Heptane/Toluene.

Solution 1 (10 wt % Hydroxyethyl methacrylate (HEMA) stock solution): 50g hydroxyethyl methacrylate monomer and 450 g water are mixed until aclear and homogeneous solution is formed.

Solution 2 (stabilizer): 1 g of Hypermer 2296 (stabilizer) is weighedout followed by the addition of 7 g of a 1:1 (vol:vol) heptane:toluene(hereafter referred to as the solvent or H/T) mixture. The stabilizersolution is stirred until a homogeneous solution is obtained.

Solution 3 (monomer phase): 0.05 g of 1,2-dihydroxy bis acrylamide(crosslinker, hereafter called DHEBA) is added to 2 g of solution 1 (10wt % HEMA stock solution). The solution is then heated to ˜40° C. tofacilitate mixing. 0.01 g of Azobisdimethyl valeronitril (radicalinitiator from Wako Chemicals, hereafter called V-65) is added to thesolution under gentle stirring.

Solution 4 (emulsion phase): Solutions 2 and 3 are mixed usingUltraturrax for 1 minute, and then sonicated for 3 minutes (6×30 secintervals) on ice (Branson digital sonifier 450 CE, 40% amplitude).Following emulsification, 0.81 g of a suspension of seed particles ofExample 2 (approximately 7 wt % dry content—hereafter referred to asseed) is added to the emulsion, and the seed-containing emulsion isallowed to swell overnight (˜16 h) at room temperature under gentlestirring/rotation). Following swelling, Solution 4 is polymerized at 50°C. for 7 h under stirring.

Example 16 (Comparative Example)

10 mL batch of acrylamide product formed in Heptane/Toluene in theabsence of seed particles.

Solution 1 (10 wt % acrylamide (AAm) stock solution): 50 g acrylamidemonomer and 450 g water are mixed until a clear and homogeneous solutionis formed.

Solution 2 (stabilizer): 1 g of Hypermer 2296 (stabilizer) is weighedout followed by the addition of 7 g of a 1:1 (vol:vol) heptane:toluene(hereafter referred to as the solvent or H/T) mixture. The stabilizersolution is stirred until a homogeneous solution is obtained.

Solution 3 (monomer phase): 0.05 g of 1,2-dihydroxy bis acrylamide(crosslinker, hereafter called DHEBA) is added to 2 g of solution 1 (10wt % AAm stock solution). The solution is then heated to ˜40° C. tofacilitate mixing. 0.08 g of Azobisdimethyl valeronitril (radicalinitiator from Wako Chemicals, hereafter called V-65) is added to thesolution under gentle stirring.

Solution 4 (emulsion phase): Solutions 2 and 3 are mixed usingUltraturrax for 1 minute, and then sonicated for 3 minutes (6×30 secintervals) on ice (Branson digital sonifier 450 CE, 40% amplitude).Following emulsification, the emulsion is allowed to swell overnight(˜16 h) at room temperature under gentle stirring/rotation). Followingswelling, Solution 4 is polymerized at 50° C. for 7 h under stirring.

Redispersion and analysis: To a 2 mL eppendorf tube 300 μL ofpolymerized emulsion, 300 μL of toluene, and 1200 μL of deionized waterwere added. The tube was vortexed and centrifuged. The supernatant wasremoved and the pellet was washed twice with 300 μL toluene anddeionized water (add to 2 mL line). Subsequently two washes in deionizedwater were performed before finally resuspending the pellet in 200 μL ofdeionized water. The product was analyzed using optical microscopy anddisc centrifuge. Microscopy was performed by diluting the redispersedproduct 10 times in 150 mM NaCl solution and image statistics wascollected from 25 images. The broad distribution made it difficult todetermine a realistic value for size using microscopy. Relative disccentrifuge diameter using CPS method 2 was determined to 1.4 μm with aCV=30%.

Example 17

Solution 1 (monomer phase): A 100 mL Duran flask equipped with a stirbar was charged with 0.9 g of N,N′-(1,2-dihydroxyethylene)bisacrylamide(DHEBA) and 39 g of H2O. The mixture was heated to ˜40° C. whilestirring until all DHEBA was dissolved, followed by the addition of 4.4g of acrylamide (AAm).

Solution 2 (stabilizer): 0.8 g of 2,2′-azobis(2-methylpropionitrile)(AIBN), 9 g of Span 80, 33 g of toluene, and 33 g of heptane were addedto a 250 mL Duran flask and shaken until all had dissolved.

Solution 3 (emulsion): 20 g of Solution 1 was added to solution 2followed by mixing using an Ultraturrax for 1 min. Furtheremulsification was performed by sonication using an UP 200s fromHielscher Ultrasound Technology with cycle setting 0.9 and amplitudesetting 40%. In the next step solution 3 and 5 g of seed particles ofExample 4) were charged into a 3-necked round bottom flask equipped witha condenser, a mechanical stirrer, and a gas inlet and stirred overnightat 100 rpm and 22° C. Finally the flask was purged with Ar (g) for 10min before heating to 70° C. while stirring at 50 rpm for 7 h.

Redispersion and analysis: To a 2 mL eppendorf tube 300 μL ofpolymerized emulsion was added and the tube was filled with1-methyl-2-pyrrolidone (NMP). The tube was vortexed and centrifuged. Thesupernatant was removed and the pellet was washed two times with NMP andtwo times with deionized water before finally resuspending the pellet in150 μL of deionized water. The product was analyzed using opticalmicroscopy and disc centrifuge. Microscopy was performed by diluting theredispersed sample 100 times in 150 mM NaCl (aq) resulting in modediameter of 1.3 μm. Characterization by disc centrifuge using CPS method2 gave a diameter of 1.5 μm with a CV=6.5% (main peak).

Example 18

Solution 1 (monomer phase): As in Example 17.

Solution 2 (stabilizer): 0.8 g of 2,2′-azodi(2-methylbutyronitrile)(AMBN), 9 g of Hypermer 2296, 64 g of dodecane were added to a 250 mLDuran flask and shaken until all had dissolved.

Solution 3 (emulsion): As in Example 17 but polymerizing for 24 h.

Redispersion and analysis: To a 2 mL eppendorf tube 300 μL ofpolymerized emulsion, 300 μL of a heptane/toluene mixture (50:50), and1200 μL of a methanol/deionized water mixture (50:50) were added. Thetube was vortexed and centrifuged. The supernatant was removed and thepellet was washed three times with 300 μL heptane/toluene mixture(50:50) and 1200 μL of a methanol/deionized water mixture (50:50).Subsequently one wash in a methanol/deionized water mixture (50:50) andtwo washes in deionized water were performed before finally resuspendingthe pellet in 100 μL of deionized water. The product was analyzed usingoptical microscopy and disc centrifuge. Microscopy was performed bydiluting the redispersed sample 100 times in 150 mM NaCl (aq) formicroscopy resulting in a mode diameter of 1.0 μm. Characterization bydisc centrifuge using CPS method 2 gave a diameter of 1.4 μm with aCV=5.2% (main peak).

Example 19

Solution 1 (monomer phase): As in Example 17.

Solution 2 (stabilizer): 0.8 g of 2,2′-azodi(2-methylbutyronitrile)(AMBN), 9 g of Abil WE09, 13 g of mineral oil, and 52 g of diethylhexylcarbonate (Tegosoft DEC) were added to a 250 mL Duran flask and shakenuntil all had dissolved.

Solution 3 (emulsion): As in Example 17.

Redispersion and analysis: As in Example 18 but final redispersion in 50μL and diluted 30 times in 150 mM NaCl (aq) for microscopy resulting ina mode diameter of 1.0 μm. Characterization by disc centrifuge using CPSmethod 2 gave a diameter of 1.7 μm with a CV=6.4% (main peak).

Example 20

Solution 1 (monomer phase): As in Example 17 but changing AAm tohydroxyethyl acrylamide (HEAAm).

Solution 2 (stabilizer): As in Example 19.

Solution 3 (emulsion): As in Example 17.

Redispersion and analysis: As in Example 18 but final redispersion in 50μL and diluted 30 times in 150 mM NaCl (aq) for microscopy resulting ina mode diameter of 1.0 μm. Characterization by disc centrifuge using CPSmethod 2 gave a diameter of 1.7 μm with a CV=6.4% (main peak).

Example 21

Solution 1 (monomer phase): A 100 mL Duran flask equipped with a stirbar was charged with 0.95 g of N,N′-(1,2-dihydroxyethylene)bisacrlamide(DHEBA) and 18 g of H2O. The mixture was heated to ˜40° C. whilestirring until all DHEBA was dissolved followed by the addition of 4.8 gof acrylamide (AAm).

Solution 2 (stabilizer): As in Example 19.

Solution 3 (emulsion): As in Example 17.

Redispersion and analysis: As in Example 18 but final redispersion in 50μL and diluted 30 times in 150 mM NaCl (aq) for microscopy resulting ina mode diameter of 1.3 μm. Characterization by disc centrifuge using CPSmethod 2 gave a diameter of 2.5 μm with a CV=5.6% (main peak).

Example 22

Solution 1 (monomer phase): As in Example 17.

Solution 2 (stabilizer): As in Example 19.

Solution 3 (emulsion): As in Example 17 but using 21 g of Solution 1 and4 g of the seed particles of Example 6.

Redispersion and analysis: As in Example 17. Microscopy was performed bydiluting the redispersed product 200 times in 150 mM NaCl (aq) resultingin a mode diameter of 1.2 μm. Characterization by disc centrifuge usingCPS method 2 gave a diameter of 2.0 μm with a CV=5.7% (main peak).

Example 23

Solution 1 (monomer phase): As in Example 17 but changing AAm to 2.5 gAAm and 0.03 g of 3-acrylamidopropionic acid (AAmPA).

Solution 2 (stabilizer): As in Example 19.

Solution 3 (emulsion): As in Example 22.

Redispersion and analysis: As in Example 17, but with final redispersionin 150 μL and diluted 200 times in 150 mM NaCl (aq) for microscopyresulting in a mode diameter of 1.3 μm. Characterization by disccentrifuge using CPS method 2 gave a diameter of 1.7 μm with a CV=8.0%(main peak).

Reaction of carboxylic acid groups: 50 μL of redispersed beads wereadded to a 2 mL eppendorf tube, washed twice with NMP and thenredispersed in 100 μL of NMP. As a first step, 40 μL of 100 mMN,N,N′,N′-tetramethyl-O—(N-succinimidyl)uronium tetrafluoroboratesolution in NMP was added and the tube placed on a thermomixer for 1 hat 1000 rpm. In a second step, 20 μL of Alexafluor 488 cadaverine (1 mgin 250 μL of NMP) was added and the tube was placed on a thermomixer for1 h at 1000 rpm. The beads were washed three times with NMP and threetimes with H2O. The beads were then redispersed in 1000 mL of 150 mMNaCl (aq) and finally diluted additionally ten times in 150 mM NaCl(aq). Imaging was done using both phase contrast and fluorescencemicroscopy confirming the presence of reactive groups. As a controlexperiment beads from Example 22 were treated with the above dyingprotocol yielding beads that were not visible in fluorescencemicroscopy.

Example 24

Solution 1 (monomer phase): As in Example 17 but with the AAm replacedwith N-(2-hydroxyethyl) methacrylamide (HEMAAm).

Solution 2 (stabilizer): As in Example 19.

Solution 3 (emulsion): As in Example 22.

Redispersion and analysis: As in Example 17 but final redispersion in150 μL and diluted 200 times in 150 mM NaCl (aq) for microscopyresulting in a mode diameter of 1.2 μm. Characterization by disccentrifuge using CPS method 2 gave a diameter of 1.7 μm with a CV=5.8%(main peak).

Example 25

Solution 1 (monomer phase): As in Example 17 but changing H₂O to 10%methanol in H₂O.

Solution 2 (stabilizer): As in Example 19.

Solution 3 (emulsion): As in Example 22.

Redispersion and analysis: As in Example 17 but final redispersion in150 μL and diluted 200 times in 150 mM NaCl (aq) for microscopyresulting in a mode diameter of 1.3 μm. Characterization by disccentrifuge using CPS method 2 gave a diameter of 1.9 μm with a CV=6.7%(main peak).

Example 26

Solution 1 (monomer phase): As in Example 17 but changing AAm tomethacrylamide (MAAm).

Solution 2 (stabilizer): As in Example 19.

Solution 3 (emulsion): As in Example 22.

Redispersion and analysis: As in Example 17 but final redispersion in100 μL and diluted 400 times in 150 mM NaCl (aq) for microscopyresulting in a mode diameter of 1.3 μm. Characterization by disccentrifuge using CPS method 2 gave a diameter of 1.8 μm with a CV=9.3%(main peak).

Example 27

Solution 1 (monomer phase): As in Example 17.

Solution 2 (stabilizer): As in Example 19.

Solution 3 (emulsion): As in Example 22 but stirring for 1 h at 20° C.instead of overnight.

Redispersion and analysis: As in Example 17 but final redispersion in100 μL and diluted 400 times in 150 mM NaCl (aq) for microscopyresulting in a mode diameter of 1.1 μm. Characterization by disccentrifuge using CPS method 2 gave a diameter of 1.7 μm with a CV=6.1%(main peak).

Example 28 (Comparative Example)

Solution 1 (monomer phase): As in Example 17.

Solution 2 (stabilizer): As in Example 19.

Solution 3 (emulsion): As in Example 17 but using 24 g of Solution 1 and1 of seed particles of Example 5.

Redispersion and analysis: As in Example 17 but final redispersion in200 μL and diluted 100 times in 150 mM NaCl (aq) for microscopyresulting in a mode diameter of 1.6 μm. Characterization by disccentrifuge using CPS method 2 gave a diameter of 3.1 μm with a CV=28%(main peak).

Example 29 (Comparative Example)

Solution 1 (monomer phase): As in Example 17.

Solution 2 (stabilizer): As in Example 19.

Solution 3 (emulsion): As in Example 17 but using seed particles ofExample 5 (not seed particles from Example 4).

Redispersion and analysis: As in Example 17 but final redispersion in200 μL and diluted 100 times in 150 mM NaCl (aq) for microscopyresulting in a mode diameter of 1.6 μm. Characterization by disccentrifuge using CPS method 2 gave a diameter of 3.1 μm with a CV=28%(main peak).

Example 30

Solution 1 (monomer phase): As in Example 17 but exchanging AAm forhydroxyethyl acrylate (HEA).

Solution 2 (stabilizer): As in Example 19.

Solution 3 (emulsion): As in Example 22.

Redispersion and analysis: As in Example 17 but final redispersion in100 μL and diluted 10 times in 150 mM NaCl (aq) for microscopy resultingin a mode diameter of 1.2 μm. Characterization by disc centrifuge usingCPS method 2 gave a diameter of 1.6 μm with a CV=6.1% (main peak).

Synthesis of Seed Particles Example 31

A 2 L jacketed round-bottom flask equipped with a mechanical anchorstirrer and a condensor was charged with 34.6 g of distilledN,N-dimethylacrylamide, 27.6 g of Kraton G1650, 1.5 g of2,2′-Azobis(2-methylpropionitrile), 747.9 g of heptane, 187.0 g oftoluene, and 1.2 g of 1-octanethiol. Once everything had dissolved thereaction mixture was purged with Ar (g) for 60 min. The reaction wasthen heated to 71° C. using a temperature controlled water bathconnected to the reactor and heated for 16 h while stirring at 400 rpm.The resulting dispersion was analyzed by dynamic light scatteringyielding a z-average diameter of 360 nm.

Highly Swellable Particles Example 32

Solution 1 (monomer phase): A 250 mL Duran flask was charged with 1.7 gof hydroxyethyl acrylamide (HEAAm), 0.02 g of acrylamidopropionic acid(AAPA), 0.26 g of N,N′-(1,2-dihydroxyethylene)bisacrylamide (DHEBA), and22.9 g of H₂O. The mixture was shaken until all had dissolved.

Solution 2 (stabilizer): 1.76 g of 2,2′-azodi(2-methylbutyronitrile)(AMBN), 24 g of Abil WE09, 26 g of mineral oil, and 104 g ofdiethylhexyl carbonate (Tegosoft DEC) were added to a 250 mL beaker andmixed with a magnetic stir bar until all had dissolved.

Solution 3 (emulsion): 71 g of solution 2 was added to solution 1followed by mixing using an Ultraturrax for 1 min. Furtheremulsification was performed by sonication using an UP 200s fromHielscher Ultrasound Technology with cycle setting 0.9 and amplitudesetting 40%. In the next step solution 3 and 4.17 g of PDMAAm seed(Example 31) were charged into a 1 L jacketed round bottom flaskequipped with a condenser, a mechanical stirrer, and a gas inlet andstirred overnight at 100 rpm at 20° C. Finally the flask was purged withAr (g) for 30 min before heating to 70° C. while stirring at 50 rpm for7 h.

Redispersion and analysis: 50 g of the crude product and 150 gN-methyl-2-pyrrolidone (NMP) were added to a 250 mL centrifugationflask. The flask was shaken for 90 min followed by centrifugation for 15min at 10000 rpm. The supernatant was removed and the product was washedadditionally once with NMP and 3 times with H₂O before finalredispersion in H₂O. The product was analyzed using optical microscopyand disc centrifuge. Microscopy was performed by diluting theredispersed product to a concentration of 0.04 wt % in 150 mM NaClsolution and image statistics was collected from 4 images yielding amode diameter of 1.3 μm. Relative disc centrifuge diameter using CPSmethod 1 was determined to 1.6 μm with a CV=8.0%.

Transfer to NMP: 20 g of particle suspension in H₂O was divided in to2×50 mL centrifugation tubes, the tubes filled with NMP and centrifugedfor 10 min at 10000 rpm. The supernatants were removed followed byadditional 3 washes in NMP before final resuspension in NMP.

Example 33

Solution 1 (monomer phase): As in Example 32.

Solution 2 (stabilizer): As in Example 32.

Solution 3 (emulsion): As in Example 32.

Redispersion and analysis: 50 g of the crude product and 150 gN-methyl-2-pyrrolidone (NMP) were added to a 250 mL centrifugationflask. The flask was shaken for 90 min followed by centrifugation for 15min at 10000 rpm. The supernatant was removed and the product was washedadditionally once with NMP and 3 times with H₂O before finalredispersion in H₂O. The product was analyzed using optical microscopyand disc centrifuge. Microscopy was performed by diluting theredispersed product to a concentration of 0.05 wt % in 150 mM NaClsolution and image statistics was collected from 25 images yielding amode diameter of 1.6 μm. Relative disc centrifuge diameter using CPSmethod 1 was determined to 1.7 μm with a CV=6.6%.

Transfer to NMP: As in Example 32.

Less Swellable Particles Example 34

Solution 1 (monomer phase): A 500 mL Duran flask was charged with 28.6 gof hydroxymethyl acrylamide (HMAAm), 0.95 g of acrylamidopropionic acid(AAPA), 19.7 g ofN,N′-((ethane-1,2-diylbis(oxy))bis(ethane-2,1-diyl))diacrylamide(EGBEAAm), and 29.4 g of H₂O. The mixture was shaken until all haddissolved.

Solution 2 (stabilizer): 3.8 g of 2,2′-azodi(2-methylbutyronitrile)(AMBN), 48 g of Abil WE09, 53 g of mineral oil, and 210 g ofdiethylhexyl carbonate (Tegosoft DEC) were added to a 600 mL beaker andmixed with a magnetic stir bar until all had dissolved.

Solution 3 (emulsion): 262 g of solution 2 was added to solution 1followed by mixing using an Ultraturrax for 1 min. Furtheremulsification was performed by sonication using an UP 200s fromHielscher Ultrasound Technology with cycle setting 0.9 and amplitudesetting 40%. In the next step solution 3 and 40 g of PDMAAm seed(Example 31) were charged into a 1 L jacketed round bottom flaskequipped with a condenser, a mechanical stirrer, and a gas inlet andstirred overnight at 100 rpm at 22° C. Finally the flask was purged withAr (g) for 30 min before heating to 70° C. while stirring at 64 rpm for7 h.

Redispersion and analysis: The crude product was divided into 2×250 mLcentrifugation flasks and centrifuged for 15 min at 5000 rpm and 10 minat 7000 rpm. The supernatants were removed and the particles wereredispersed in isopropanol. The redispersed particles were then splitinto a total of 6×250 mL centrifuge bottles, washed 4 times withisopropanol and 6 times with H₂O and before final redispersion in H₂O.The particles were then filtered through three different filtration meshfabrics: Sefar Nitex 03-64/32, Sefar Nitex 03-30/18, and Sefar Nitex03-1/1. The product was analyzed using optical microscopy and disccentrifuge. Bright field microscopy was performed by diluting theredispersed product to a concentration of 0.01 wt % in 150 mM NaClsolution and image statistics was collected from 18 images yielding amode diameter of 0.91 μm. Relative disc centrifuge diameter using CPSmethod 3 was determined to 0.54 μm with a CV=6.2%.

Example 35

Solution 1 (monomer phase): A 500 mL Duran flask was charged with 27.0 gof hydroxymethyl acrylamide (HMAAm), 2.6 g of acrylamidopropionic acid(AAPA), 19.7 g ofN,N′-((ethane-1,2-diylbis(oxy))bis(ethane-2,1-diyl))diacrylamide(EGBEAAm), and 49.3 g of H₂O. The mixture was shaken until all haddissolved.

Solution 2 (stabilizer): As in Example 34.

Solution 3 (emulsion): As in Example 34.

Redispersion and analysis: The crude product was divided into 2×250 mLcentrifugation flasks and centrifuged for 25 min at 5000 rpm. Thesupernatants were removed and the particles were redispersed inisopropanol. The redispersed particles were then split into a total of6×250 mL centrifuge bottles, washed 4 times with isopropanol and 5 timeswith H₂O and before final redispersion in H₂O. The particles were thenfiltered through two different filtration mesh fabrics: Sefar Nitex03-30/18, and Sefar Nitex 03-1/1. The product was analyzed using opticalmicroscopy and disc centrifuge. Bright field microscopy was performed bydiluting the redispersed product to a concentration 0.01 wt % in 150 mMNaCl solution and image statistics was collected from 21 images yieldinga mode diameter of 0.90 μm. Relative disc centrifuge diameter using CPSmethod 3 was determined to 0.53 μm with a CV=5.2%.

Example 36

Solution 1 (monomer phase): A 500 mL Duran flask was charged with 24.8 gof hydroxymethyl acrylamide (HMAAm), 5.0 g of acrylamidopropionic acid(AAPA), 19.9 g ofN,N′-((ethane-1,2-diylbis(oxy))bis(ethane-2,1-diyl))diacrylamide(EGBEAAm), and 49.7 g of H₂O. The mixture was shaken until all haddissolved.

Solution 2 (stabilizer): 3.8 g of 2,2′-azodi(2-methylbutyronitrile)(AMBN), 52 g of Abil WE09, 57 g of mineral oil, and 228 g ofdiethylhexyl carbonate (Tegosoft DEC) were added to a 1000 mL beaker andmixed with a magnetic stir bar until all had dissolved.

Solution 3 (emulsion): 284 g of solution 2 was added to solution 1followed by mixing using an Ultraturrax for 1 min. Furtheremulsification was performed by sonication using an UP 200s fromHielscher Ultrasound Technology with cycle setting 0.9 and amplitudesetting 40%. In the next step solution 3 and 17 g of PDMAAm seed(Example 31) were charged into a 1 L jacketed round bottom flaskequipped with a condenser, a mechanical stirrer, and a gas inlet andstirred overnight at 100 rpm at 25° C. Finally the flask was purged withAr (g) for 30 min before heating to 70° C. while stirring at 62 rpm for7 h.

Redispersion and analysis: The crude product was divided into 2×250 mLcentrifugation flasks and centrifuged for 20 min at 3000 rpm and 10 min5000 rpm. The supernatants were removed and the particles wereredispersed in isopropanol. The redispersed particles were then splitinto a total of 6×250 mL centrifuge bottles, washed 4 times withisopropanol and 6 times with H₂O and before final redispersion in H₂O.The particles were then filtered through two different filtration meshfabrics: Sefar Nitex 03-30/18, and Sefar Nitex 03-1/1. The product wasanalyzed using optical microscopy and disc centrifuge. Bright fieldmicroscopy was performed by diluting the redispersed product to aconcentration of 0.01 wt % in 150 mM NaCl solution and image statisticswas collected from 30 images yielding a mode diameter of 1.0 μm.Relative disc centrifuge diameter using CPS method 3 was determined to0.57 μm with a CV=6.3%.

Example 37

Solution 1 (monomer phase): A 250 mL Duran flask was charged with 1.8 gof hydroxymethyl acrylamide (HMAAm), 0.7 g of acrylamidopropionic acid(AAPA), 4.9 g ofN,N′-((ethane-1,2-diylbis(oxy))bis(ethane-2,1-diyl))diacrylamide(EGBEAAm), and 17.3 g of H₂O. The mixture was shaken until all haddissolved.

Solution 2 (stabilizer): 2.8 g of 2,2′-azodi(2-methylbutyronitrile)(AMBN), 35 g of Abil WE09, 38 g of mineral oil, and 153 g ofdiethylhexyl carbonate (Tegosoft DEC) were added to a 400 mL beaker andmixed with a magnetic stir bar until all had dissolved.

Solution 3 (emulsion): 66 g of solution 2 was added to solution 1followed by mixing using an Ultraturrax for 1 min. Furtheremulsification was performed by sonication using an UP 200s fromHielscher Ultrasound Technology with cycle setting 0.9 and amplitudesetting 40%. In the next step solution 3 and 9.9 g of PDMAAm seed(Example 31) were charged into a 1 L jacketed round bottom flaskequipped with a condenser, a mechanical stirrer, and a gas inlet andstirred overnight at 100 rpm at 19° C. Finally the flask was purged withAr (g) for 30 min before heating to 70° C. while stirring at 55 rpm for7 h.

Redispersion and analysis: The crude product added to a 250 mLcentrifugation flask and centrifuged for 20 min at 5000 rpm. Thesupernatants were removed and the particles were redispersed inisopropanol. The redispersed particles were then split into a total of2×250 mL centrifuge bottles, washed 3 times with isopropanol and 6 timeswith H₂O and before final redispersion in H₂O. The particles were thenfiltered through two different filtration mesh fabrics: Sefar Nitex03-30/18, and Sefar Nitex 03-1/1. The product was analyzed using opticalmicroscopy and disc centrifuge. Bright field microscopy was performed bydiluting the redispersed product to a concentration of 0.01 wt % in 150mM NaCl solution and image statistics was collected from 21 imagesyielding a mode diameter of 0.84 μm. Relative disc centrifuge diameterusing CPS method 3 was determined to 0.41 μm with a CV=9.5%.

Example 38

Solution 1 (monomer phase): A 250 mL Duran flask was charged with 12.3 gof N,N′-((ethane-1,2-diylbis(oxy))bis(ethane-2,1-diyl))diacrylamide(EGBEAAm), and 12.4 g of H₂O. The mixture was shaken until all haddissolved.

Solution 2 (stabilizer): As in Example 37.

Solution 3 (emulsion): As in Example 37.

Redispersion and analysis: As in Example 37. The product was analyzedusing optical microscopy and disc centrifuge. Bright field microscopywas performed by diluting the redispersed product to a concentration of0.01 wt % in 150 mM NaCl solution and image statistics was collectedfrom 24 images yielding a mode diameter of 0.85 μm. Relative disccentrifuge diameter using CPS method 3 was determined to 0.50 μm with aCV=8.7%.

Example 39

Solution 1 (monomer phase): A 250 mL Duran flask was charged with 2.1 gof hydroxymethyl acrylamide (HMAAm), 0.3 g of acrylic acid (AA), 4.9 gof N,N′-((ethane-1,2-diylbis(oxy))bis(ethane-2,1-diyl))diacrylamide(EGBEAAm), and 17.3 g of H₂O. The mixture was shaken until all haddissolved.

Solution 2 (stabilizer): As in Example 37.

Solution 3 (emulsion): As in Example 37.

Redispersion and analysis: As in Example 37. The product was analyzedusing optical microscopy and disc centrifuge. Bright field microscopywas performed by diluting the redispersed product to a concentration of0.01 wt % in 150 mM NaCl solution and image statistics was collectedfrom 21 images yielding a mode diameter of 0.85 μm. Relative disccentrifuge diameter using CPS method 3 was determined to 0.42 μm with aCV=7.6%.

2-Stage Particles Example 40 Stage 1: Swelling and Polymerization ofParticles in the Absence of Crosslinker

Solution 1 (12 wt % Hydroxymethyl acrylamide (HMAAm) stock solution):125 g hydroxymethyl acrylamide monomer (48% solution) and 375 g DI waterwere mixed until a clear and homogeneous solution is formed.

Solution 2 (monomer phase): 5 μL of 1-thioglycerol was added to 2 g ofsolution 1 (25 wt % HMAAm stock solution). The solution was then heatedto ˜40° C. to facilitate mixing. 0.08 g of azobisdimethyl valeronitril(V-65) was added to the solution under gentle stirring.

Solution 3 (stabilizer): 1 g of Hypermer 2296 (stabilizer) was weighedout followed by the addition of 7 g of a 1:1 (vol:vol) heptane:toluene(hereafter referred to as the solvent or H/T) mixture. The stabilizersolution was stirred until a homogeneous solution was obtained.

Solution 4 (emulsion phase): Solutions 2 and 3 were mixed usingUltraturrax for 1 minute, and then sonicated for 3 minutes (6×30 secintervals) on ice (Branson digital sonifier 450 CE, 40% amplitude).Following emulsification, 0.81 g of a PDMAAm seed (Example 3) was addedto the emulsion, and the seed-containing emulsion was allowed to swellovernight (˜16 h) at room temperature under gentle stirring/rotation).Following swelling, Solution 4 was polymerized at 50° C. for 7 h understirring.

Stage 2: Secondary Swelling and Polymerization of Cross-Linked Particles

Solution 5 (second stabilizer solution): Same as Solution 3.

Solution 6 (second monomer phase): 0.05 g of 1,2-dihydroxy bisacrylamide (DHEBA) was added to 8 mL of solution 1 (25 wt % HMAAm stocksolution). The solution was then heated to ˜40° C. to facilitate mixing.0.05 g of azobisdimethyl valeronitril (V-65) was added to the solutionunder gentle stirring.

Solution 7 (second emulsion phase): Solutions 5 and 6 were mixed usingUltraturrax for 1 minute, and then sonicated for 3 minutes (6×30 secintervals) on ice (Branson digital sonifier 450 CE, 40% amplitude).

Redispersion and analysis: To a 2 mL eppendorf tube 300 μL ofpolymerized emulsion, 300 μL of a heptane/toluene mixture (50:50), and1200 μL of a methanol/deionized water mixture (50:50) were added. Thetube was vortexed and centrifuged. The supernatant was removed and thepellet was washed three times with 300 μL heptane/toluene mixture(50:50) and 1200 μL of a methanol/deionized water mixture (50:50).Subsequently one wash in a methanol/deionized water mixture (50:50) andtwo washes in deionized water were performed before finally resuspendingthe pellet in 150 μL of deionized water. The product was analyzed usingoptical microscopy and disc centrifuge. Bright field microscopy wasperformed by diluting the redispersed product 10 times in 150 mM NaClsolution and image statistics was collected from 21 images yielding amode diameter of 4.0 μm. Relative disc centrifuge diameter using CPSmethod 3 was determined to 0.69 μm with a CV=3.4%.

Example 41 Stage 1: Swelling and Polymerization of Particles in theAbsence of Crosslinker

Solution 1 (24 wt % Hydroxymethyl acrylamide (HMAAm) stock solution): 25g hydroxymethyl acrylamide monomer (48% solution) and 25 g DI water aremixed until a clear and homogeneous solution is formed.

Solution 2 (monomer phase): As in Example 40, but using 20 μL of1-thioglycerol.

Solution 3 (stabilizer): As in Example 40.

Solution 4 (emulsion phase): As in Example 40.

Stage 2: Secondary Swelling and Polymerization of Cross-Linked Particles

As in Example 40, but using the 50 wt % HMAAm stock solution.

Redispersion and analysis: To a 2 mL eppendorf tube 300 μL ofpolymerized emulsion, 300 μL of a heptane/toluene mixture (50:50), and1200 μL of a methanol/deionized water mixture (50:50) were added. Thetube was vortexed and centrifuged. The supernatant was removed and thepellet was washed three times with 300 μL heptane/toluene mixture(50:50) and 1200 μL of a methanol/deionized water mixture (50:50).Subsequently one wash in a methanol/deionized water mixture (50:50) andtwo washes in deionized water were performed before finally resuspendingthe pellet in 200 μL of deionized water. The product was analyzed usingoptical microscopy and disc centrifuge. Bright field microscopy wasperformed by diluting the redispersed product 10 times in 150 mM NaClsolution and image statistics was collected from 21 images yielding amode diameter of 3.4 μm. Relative disc centrifuge diameter using CPSmethod 3 was determined to 0.89 μm with a CV=3.8%.

Example 42 Stage 1: Swelling and Polymerization of Particles in theAbsence of Crosslinker

Solution 1 (monomer phase): A 250 mL Duran flask was charged with 16.0 gof hydroxyethyl acrylamide (HEAAm), 0.3 g of 1-thioglycerol, and 8.3 gof H₂O. The mixture was shaken until all had dissolved.

Solution 2 (stabilizer): 2.6 g of 2,2′-azodi(2-methylbutyronitrile)(AMBN), 32 g of Abil WE09, 35 g of mineral oil, and 139 g ofdiethylhexyl carbonate (Tegosoft DEC) were added to a 250 mL beaker andmixed with a magnetic stir bar until all had dissolved.

Solution 3 (emulsion): 65 g of solution 2 was added to solution 1followed by mixing using an Ultraturrax for 1 min. Furtheremulsification was performed by sonication using an UP 200s fromHielscher Ultrasound Technology with cycle setting 0.9 and amplitudesetting 40%. In the next step solution 3 and 10.3 g of PDMAAm seed werecharged into a 250 mL 3-necked round bottom flask equipped with acondenser, a mechanical stirrer, and a gas inlet and stirred overnightat 100 rpm at 20° C. Finally the flask was purged with Ar (g) for 30 minbefore heating to 70° C. while stirring at 53 rpm for 7 h.

Stage 2: Secondary Swelling and Polymerization of Cross-Linked Particles

Solution 5 (second monomer phase): A 250 mL Duran flask was charged with7.1 g of hydroxyethyl acrylamide (HEAAm), 2.4 g ofN,N′-((ethane-1,2-diylbis(oxy))bis(ethane-2,1-diyl))diacrylamide(EGBEAAm), and 14.2 g of H₂O. The mixture was shaken until all haddissolved.

Solution 6 (second stabilizer): 2.6 g of2,2′-azodi(2-methylbutyronitrile) (AMBN), 34 g of Abil WE09, 37 g ofmineral oil, and 147 g of diethylhexyl carbonate (Tegosoft DEC) wereadded to a 400 mL beaker and mixed with a magnetic stir bar until allhad dissolved.

Solution 7 (second emulsion): 69 g of solution 6 was added to solution 5followed by mixing using an Ultraturrax for 1 min. Furtheremulsification was performed by sonication using an UP 200s fromHielscher Ultrasound Technology with cycle setting 0.9 and amplitudesetting 40%. In the next step solution 7 and 7.7 g of polymerizedsolution 3 were charged into a 250 mL 3-necked round bottom flaskequipped with a condenser, a mechanical stirrer, and a gas inlet andstirred overnight at 100 rpm at 22° C. Finally the flask was purged withAr (g) for 30 min before heating to 70° C. while stirring at 56 rpm for7 h.

Redispersion and analysis: The crude product added to a 250 mLcentrifugation flask and centrifuged for 30 min at 5000 rpm. Thesupernatant was removed and the particles were redispersed inisopropanol. The redispersed particles were then washed 2 times withisopropanol and 14 times with H₂O and before final redispersion in H₂O.The product was analyzed using optical microscopy and disc centrifuge.Bright field microscopy was performed by diluting the redispersedproduct to a concentration of 0.05 wt % in 150 mM NaCl solution andimage statistics was collected from 97 images yielding a mode diameterof 5.6 μm. Relative disc centrifuge diameter using CPS method 3 wasdetermined to 2.0 μm with a CV=7.5%.

Example 43 Stage 1: Swelling and Polymerization of Particles in theAbsence of Crosslinker

Solution 1 (monomer phase): As in Example 42.

Solution 2 (stabilizer): 2.6 g of 2,2′-azodi(2-methylbutyronitrile)(AMBN), 32 g of Hypermer 2296, 88 g of toluene, and 89 g of heptane wereadded to a 400 mL beaker and mixed with a magnetic stir bar until allhad dissolved.

Solution 3 (emulsion): As in Example 42, but using this example'ssolutions.

Stage 2: Secondary Swelling and Polymerization of Cross-Linked Particles

Solution 5 (second monomer phase): A 250 mL Duran flask was charged with7.0 g of hydroxyethyl acrylamide (HEAAm), 2.3 g ofN,N′-((ethane-1,2-diylbis(oxy))bis(ethane-2,1-diyl))diacrylamide(EGBEAAm), and 14.1 g of H₂O. The mixture was shaken until all haddissolved.

Solution 6 (second stabilizer): 0.96 g of2,2′-azodi(2-methylbutyronitrile) (AMBN), 12 g of Hypermer 2296, 34 g oftoluene, and 34 g of heptane were added to a 250 mL beaker and mixedwith a magnetic stir bar until all had dissolved.

Solution 7 (second emulsion): 67 g of solution 6 was added to solution 5followed by mixing using an Ultraturrax for 1 min. Furtheremulsification was performed by sonication using an UP 200s fromHielscher Ultrasound Technology with cycle setting 0.9 and amplitudesetting 40%. In the next step solution 7 and 7.7 g of polymerizedsolution 3 were charged into a 250 mL 3-necked round bottom flaskequipped with a condenser, a mechanical stirrer, and a gas inlet andstirred overnight at 100 rpm at 22° C. Finally the flask was purged withAr (g) for 30 min before heating to 70° C. while stirring at 55 rpm for7 h.

Redispersion and analysis: The crude product added to a 250 mLcentrifugation flask and centrifuged for 5 min at 3000 rpm. Thesupernatant was removed and the particles were redispersed inisopropanol. The redispersed particles were then washed 3 times withisopropanol and 10 times with H₂O and before final redispersion in H₂O.The product was analyzed using optical microscopy and disc centrifuge.Bright field microscopy was performed by diluting the redispersedproduct to a concentration of 0.02 wt % in 150 mM NaCl solution andimage statistics was collected from 72 images yielding a mode diameterof 4.4 μm. Relative disc centrifuge diameter using CPS method 3 wasdetermined to 1.3 μm with a CV=7.1%.

Multifunctional Cross-Linkers Example 44

Synthesis of N,N-bis(2-acrylamidoethyl)acrylamide (BAAmEAAm).Diethylenetriamine (14.63 g; 142 mmol) was dissolved in 200 mLacetonitrile. Potassium carbonate (64.68 g; 468 mmol) was added and thereaction mixture was cooled to 0° C. with an ice bath. Acryloyl chloride(36.3 mL, 447 mmol) was added dropwise over approximately 45 minutes.The ice bath was removed and the reaction was stirred at ambienttemperature for two hours. The reaction mixture was filtered and thefiltercake was rinsed with 200 mL CH₂Cl₂. Inhibitor (phenothiazine, 10.1mg) was added to the filtrate before the volatiles were removed underreduced pressure at 25° C. The crude material was purified by dry flashchromatography on silica using a gradient of methanol indichloromethane. The fractions containing product were pooled and 9.9 mgof phenothiazine was added before finally drying the product undervacuum. Yield 12.97 g.

Example 45

Solution 1 (monomer phase): A 250 mL Duran flask was charged with 3.1 gof hydroxyethyl acrylamide (HEAAm), 0.7 g of acrylamidopropionic acid(AAPA), 3.7 g ofN,N′-((ethane-1,2-diylbis(oxy))bis(ethane-2,1-diyl))diacrylamide(EGBEAAm), 0.3 g of N,N-bis(2-acrylamidoethyl)acrylamide (BAAmEAAm) and17 g of H₂O. The mixture was shaken until all had dissolved.

Solution 2 (stabilizer): 2.6 g of 2,2′-azodi(2-methylbutyronitrile)(AMBN), 32 g of Abil WE09, 35 g of mineral oil, and 140 g ofdiethylhexyl carbonate (Tegosoft DEC) were added to a 250 mL beaker andmixed with a magnetic stir bar until all had dissolved.

Solution 3 (emulsion): 65 g of solution 2 was added to solution 1followed by mixing using an Ultraturrax for 1 min. Furtheremulsification was performed by sonication using an UP 200s fromHielscher Ultrasound Technology with cycle setting 0.9 and amplitudesetting 40%. In the next step solution 3 and 9.87 g of PDMAAm seed(Example 31) were charged into a 250 mL 3-necked round bottom flaskequipped with a condenser, a mechanical stirrer, and a gas inlet andstirred overnight at 100 rpm at 20° C. Finally the flask was purged withAr (g) for 30 min before heating to 70° C. while stirring at 53 rpm for7 h.

Redispersion and analysis: 70 g the crude product was added into a 1000mL centrifugation flask together with 500 mL H₂O and 2-butanol followedby shaking for 30 min. The mixture was then centrifuged and the top2-butanol phase removed followed by additionally 2×200 mL 2-butanolwashes. The suspension was then divided into 6×250 mL centrifuge bottleand washed with H₂O 6 times before final redispersion in H₂O. Theproduct was analyzed using optical microscopy and disc centrifuge.Bright field microscopy was performed by diluting the redispersedproduct to a concentration of 0.01 wt % in 150 mM NaCl solution andimage statistics was collected from 25 images yielding a mode diameterof 0.82 μm. Relative disc centrifuge diameter using CPS method 3 wasdetermined to 0.43 μm with a CV=4.6%.

Example 46

Solution 1 (monomer phase): A 250 mL Duran flask was charged with 3.0 gof hydroxyethyl acrylamide (HEAAm), 0.7 g of acrylamidopropionic acid(AAPA), 3.7 g ofN,N′-((ethane-1,2-diylbis(oxy))bis(ethane-2,1-diyl))diacrylamide(EGBEAAm), 0.2 g of 4-arm PEG-acrylamide and 17 g of H₂O. The mixturewas shaken until all had dissolved.

Solution 2 (stabilizer): As in Example 45.

Solution 3 (emulsion): As in Example 45.

Redispersion and analysis: 70 g the crude product was added into a 1000mL centrifugation flask together with 500 mL H₂O and 2-butanol followedby shaking for 60 min. The mixture was then centrifuged and the top2-butanol phase removed followed by additionally one wash with2-butanol. The suspension was then washed with H₂O 5 times before finalredispersion in H₂O. The product was analyzed using optical microscopyand disc centrifuge. Bright field microscopy was performed by dilutingthe redispersed product to a concentration of 0.01 wt % in 150 mM NaClsolution and image statistics was collected from 11 images yielding amode diameter of 0.86 μm. Relative disc centrifuge diameter using CPSmethod 3 was determined to 0.41 μm with a CV=6.0%.

Example 47

Solution 1 (monomer phase): A 250 mL Duran flask was charged with 1.8 gof hydroxyethyl acrylamide (HEAAm), 0.7 g of acrylamidopropionic acid(AAPA), 2.5 g of N,N-bis(2-acrylamidoethyl)acrylamide (BAAmEAAm) and 20g of H₂O. The mixture was shaken until all had dissolved.

Solution 2 (stabilizer): As in Example 45.

Solution 3 (emulsion): As in Example 45.

Redispersion and analysis: 70 g the crude product was added into a 1000mL centrifugation flask together with 500 mL H₂O and 2-butanol followedby shaking for 60 min. The mixture was then centrifuged and the top2-butanol phase removed followed by additionally one wash with2-butanol. The suspension was then divided into 6×250 mL centrifugebottle and washed with H₂O 5 times before final redispersion in H₂O. Theproduct was analyzed using optical microscopy and disc centrifuge.Bright field microscopy was performed by diluting the redispersedproduct to a concentration of 0.01 wt % in 150 mM NaCl solution andimage statistics was collected from 24 images yielding a mode diameterof 0.67 μm. Relative disc centrifuge diameter using CPS method 3 wasdetermined to 0.39 μm with a CV=6.4%.

Activation of a Hydrogel Containing Carboxylate Groups. Example 48

To a 7200 μL suspension of particles of Example 32 in NMP, is added 22.6mg of O—(N-Succinimidyl)-N,N,N′,N′-tetramethyluroniumtetrafluoroborate(TSTU), 27 μL of tributylamine, and NMP to a final volume of 7.5 mL.After shaking at room temperature for 1 h, the suspension is centrifugedat 15,000 rpm for 50 min. The supernatant is removed from the resultingpellet, and the pellet is re-suspended into 4 mL of fresh NMP andcentrifuged for 1 h at 15,000 rpm. The supernatant is removed from theNHS ester activated hydrogel comprising resulting pellet. Repeat the 4mL NMP wash/spin one more time.

Example 49

As per Example 48, but using a 7300 μL suspension of particles ofExample 33.

Conjugation of the Hydrogel with Amine Terminal DNA Probe

Example 50

An amount of 193.4 μL of a 5.17 mM NMP solution of 5′-amine terminated30-mer oligonucleotide (that has tetrabutylammonium counter ion for eacholigonucleotide phosphate group) is added to the carboxylate NHS esterhydrogel pellet from Example 48. This suspension is diluted to 9.976 mLwith NMP. After addition of 23.8 microliter of 42 mM tributylamine inNMP, total reaction volume of 10 mL, the reaction vessel is agitated ina thermomixer at 66° C. for 16 h. It is added 10 mL water and cooled toroom temperature, to this mixture is added 10 mL 0.25 M NaOH. It isagitated in a thermomixer for 15 min at room temperature and 1 mL 50×TEis added, followed by centrifugation for 50 min, and removal of thesupernatant. The resulting pellet is re-suspended in 10 milliliter of1×TE, followed by centrifugation for 50 min, and removal of thesupernatant. At this point, the hydrogel pellet is re-suspended in 10 mLof 1×TE buffer and heated at 80° C. for 1 h; after centrifugation (50min at 15,000 rpm) and removal of the supernatant, the conjugatedhydrogels are washed, as above, with 10 mL of 1×TE, followed bycentrifugation for 50 min, and removal of the supernatant. The resultingpellet is re-suspended in 14 mL of de-ionized water and filtered with 5μm syringe filter for later use

Example 51

As in Example 50, but using the carboxylate NHS ester hydrogel pelletfrom Example 49.

Templating with OCP143 Library, Chip Loading and Sequencing

Example 52

For more information see user guides on thermofisher.com, the followingis a brief description. The conjugated particles were templated with aDNA library of size 100-120 basepairs by emulsion PCR. The templatingwas performed on the automated Ion Chef™ instrument using 530 v1standard Ion Chef™ reagents and consumables. The particles were manuallyloaded onto the chips and the sequencing was performed on an Ion Proton™sequencer. Key metrics from the sequencing from conjugated particles inExample 50: 1.4 M total reads, 98.6% raw read accuracy and 75 bases AQ20mean (probability of incorrect base call 1%). Key metrics from thesequencing from conjugated particles in Example 51: 3.0 M total reads,98.7% raw read accuracy and 83 bases AQ20 mean

1.-122. (canceled)
 123. A method of nucleic acid amplification,comprising: providing monodisperse cross-linked hydrogel polymerparticles, wherein each particle comprises a polymer formed from (a) ahydrophilic vinylic monomer having a log P_(oct/wat) (log P) of lessthan about 1, and (b) a crosslinker comprising at least two vinylgroups, wherein each particle comprises one or more oligonucleotidesattached to the particle; and amplifying the one or moreoligonucleotides by polymerase chain reaction (PCR) or emulsion PCRamplification to provide a plurality of polynucleotide templatesattached to the particle.
 124. The method of claim 123, wherein thehydrophilic vinylic monomer is selected from an acrylamide monomer, anacrylate monomer, a methacrylamide monomer, a methacrylic monomer, and amonomer comprising at least one compound of formula (I):

wherein: R¹ is —H, —CH₃ or —CH₂CH₃; R² is —OR³ or —N(R⁴)R⁵; R³ is —H,—C₁-C₆ alkyl, or —C₁-C₆ alcohol; and R⁴ and R⁵ are each independentlyselected from —H, —C₁-C₆ alkyl, —C₁-C₆ haloalkyl, —C₁-C₆ alcohol,wherein R³ and/or R⁴ and/or R^(S) are optionally each independentlysubstituted, where chemically possible, by 1 to 5 substituents which areeach independently at each occurrence selected from: oxo, ═NR^(a),═NOR^(a), halo, nitro, cyano, NR^(a)R^(a), NR^(a)S(O)₂R^(a),NR^(a)CONR^(a)R^(a), NR^(a)CO₂R^(a), OR^(a); SR^(a), S(O)R^(a),S(O)₂OR^(a), S(O)₂R^(a), S(O)₂NR^(a)R^(a), CO₂R^(a) C(O)R^(a),CONR^(a)R^(a), C₁-C₄-alkyl, C₂-C₄-alkenyl, C₂-C₄-alkynyl, C₁-C₄haloalkyl; wherein R^(a) is independently at each occurrence selectedfrom: H and C₁-C₄ alkyl.
 125. The method of claim 123, wherein thecrosslinker comprises at least one compound of formula (IIa) or (IIb):

wherein R⁶ is selected from —C₁-C₆ alkyl-, —C₁-C₆ heteroalkyl-, —C₁-C₆cycloalkyl-, —C₁-C₆ hydroxyalkyl-, —C₁-C₆ ether-, or polyethercomprising 2 to 100 C₂-C₃ ether units; R⁷ and Rare each independentlyselected from —H, —C₁-C₆ alkyl, —C₁-C₆ heteroalkyl, —C₃-C₆ cycloalkyl,—C₁-C₆ hydroxyalkyl, or —C₁-C₆ ether; R⁹ is —N(R¹¹)C(O)CH═CH₂; R¹⁰ isselected from —H and —N(R¹²)C(O)CH═CH₂; and R¹¹ and R¹² are eachindependently selected from —H, —C₁-C₆ alkyl, —C₁-C₆ heteroalkyl, —C₃-C₆cycloalkyl, —C₁-C₆ hydroxyalkyl, or —C₁-C₆ ether; optionally wherein oneor more of R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹ and R¹² are independentlysubstituted, where chemically possible, by 1 to 5 substituents which areeach independently at each occurrence selected from: oxo, ═NR^(a),═NOR^(a), halo, nitro, cyano, NR^(a)R^(a), NR^(a)S(O)₂R^(a),NR^(a)CONR^(a)R^(a), NR^(a)CO₂R^(a), OR^(a); SR^(a), S(O)R^(a),S(O)₂OR^(a), S(O)₂R^(a), S(O)₂NR^(a)R^(a), CO₂R^(a) C(O)R^(a),CONR^(a)R^(a), C₁-C₄-alkyl, C₂-C₄-alkenyl, C₂-C₄-alkynyl, C₁-C₄haloalkyl; wherein R^(a) is independently at each occurrence selectedfrom: H, C₁-C₄ alkyl and C₁-C₄ alkenyl.
 126. The method of claim 123,wherein the crosslinker is or comprisesN,N′-(1,2-dihydroxyethylene)bisacrylamide,N,N′-methylenebis(acrylamide), N,N′-ethylenebis(acrylamide), glycerol1,3-diglycerolate diacrylate, piperazine diacrylamide,N,N′-((ethane-1,2-diylbis(oxy))bis(ethane-2,1-diyl))diacrylamide,polyethyleneglycol diacrylamide (MW≤2000), 4-Arm PEG-Acrylamide(MW≤2000), N,N-bis(2-acrylamidoethyl)acrylamide, 1,2-dihydroxybis-acrylamide,N,N′-((ethane-1,2-diylbis(oxy))bis(ethane-2,1-diyl))diacrylamide or acombination thereof.
 127. The method of claim 123, wherein the particleseach absorb at least 20% of their weight of the aqueous solution.
 128. Amethod for nucleic acid amplification, comprising: providingmonodisperse cross-linked hydrogel polymer particles, wherein eachparticle comprises a polymer formed from (a) a hydrophilic vinylicmonomer having a log P_(oct/wat) (log P) of less than about 1, and (b) acrosslinker comprising at least two vinyl groups, wherein each particlecomprises one or more oligonucleotides attached to the particle;hybridizing a polynucleotide to at least one of the one or more firstoligonucleotides; and conducting a primer extension reaction on thepolynucleotide.
 129. The method of claim 128, wherein the methodcomprises: (a) wherein the one or more first oligonucleotides aresingle-stranded oligonucleotide; (b) wherein the polynucleotide is asingle-stranded template polynucleotide; (c) hybridizing thesingle-stranded oligonucleotide to the single-stranded templatepolynucleotide; and (d) contacting the single-stranded templatepolynucleotide with a polymerase and at least one nucleotide underconditions suitable for the polymerase to catalyze polymerization of atleast one nucleotide onto the single-stranded oligonucleotide so as togenerate an extended single-stranded oligonucleotide.
 130. The method ofclaim 129, wherein the method further comprises: (e) removing thesingle-stranded template polynucleotide from the extendedsingle-stranded oligonucleotide so that the single-strandedoligonucleotide remains attached to the polymeric particle; (f)hybridizing the remaining single-stranded oligonucleotide to a primer;(g) contacting the remaining single-stranded oligonucleotide with asecond polymerase and a second at least one nucleotide, under conditionssuitable for the second polymerase to catalyze polymerization of thesecond at least one nucleotide onto the primer so as to generate anextended primer; and (h) detecting the addition of the second nucleotideto determine the sequence of the subsequent extended single-strandedoligonucleotide.
 131. The method of claim 130, wherein detecting thenucleotide addition comprises detecting fluorescent emission from theadded second nucleotide, wherein the second nucleotide is fluorescent,or detecting a pH change in the local environment of the polymericparticle with an ionic sensor.
 132. The method of claim 131, wherein theionic sensor is a field effect transistor (FET).
 133. The method ofclaim 129, wherein the hydrophilic vinylic monomer is selected from anacrylamide monomer, an acrylate monomer, a methacrylamide monomer, amethacrylic monomer, and a monomer comprising at least one compound offormula (I):

wherein: R¹ is —H, —CH₃ or —CH₂CH₃; R² is —OR³ or —N(R⁴)R⁵; R³ is —H,—C₁-C₆ alkyl, or —C₁-C₆ alcohol; and R⁴ and R⁵ are each independentlyselected from —H, —C₁-C₆ alkyl, —C₁-C₆ haloalkyl, —C₁-C₆ alcohol,wherein R³ and/or R⁴ and/or R⁵ are optionally each independentlysubstituted, where chemically possible, by 1 to 5 substituents which areeach independently at each occurrence selected from: oxo, ═NR^(a),═NOR^(a), halo, nitro, cyano, NR^(a)R^(a), NR^(a)S(O)₂R^(a),NR^(a)CONR^(a)R^(a), NR^(a)CO₂R^(a), OR^(a); SR^(a), S(O)R^(a),S(O)₂OR^(a), S(O)₂R^(a), S(O)₂NR^(a)R^(a), CO₂R^(a) C(O)R^(a),CONR^(a)R^(a), C₁-C₄-alkyl, C₂-C₄-alkenyl, C₂-C₄-alkynyl, C₁-C₄haloalkyl; wherein R^(a) is independently at each occurrence selectedfrom: H and C₁-C₄ alkyl.
 134. The method of claim 129, wherein thecrosslinker comprises at least one compound of formula (IIa) or (IIb):

wherein R⁶ is selected from —C₁-C₆ alkyl-, —C₁-C₆ heteroalkyl-, —C₁-C₆cycloalkyl-, —C₁-C₆ hydroxyalkyl-, —C₁-C₆ ether-, or polyethercomprising 2 to 100 C₂-C₃ ether units; R⁷ and R⁸ are each independentlyselected from —H, —C₁-C₆ alkyl, —C₁-C₆ heteroalkyl, —C₃-C₆ cycloalkyl,—C₁-C₆ hydroxyalkyl, or —C₁-C₆ ether; R⁹ is —N(R¹¹)C(O)CH═CH₂; R¹⁰ isselected from —H and —N(R¹²)C(O)CH═CH₂; and R¹¹ and R¹² are eachindependently selected from —H, —C₁-C₆ alkyl, —C₁-C₆ heteroalkyl, —C₃-C₆cycloalkyl, —C₁-C₆ hydroxyalkyl, or —C₁-C₆ ether; optionally wherein oneor more of R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹ and R¹² are independentlysubstituted, where chemically possible, by 1 to 5 substituents which areeach independently at each occurrence selected from: oxo, ═NR^(a),═NOR^(a), halo, nitro, cyano, NR^(a)R^(a), NR^(a)S(O)₂R^(a),NR^(a)CONR^(a)R^(a), NR^(a)CO₂R^(a), OR^(a); SR, S(O)R^(a), S(O)₂OR^(a),S(O)₂R^(a), S(O)₂NR^(a)R^(a), CO₂R^(a) C(O)R^(a), CONR^(a)R^(a),C₁-C₄-alkyl, C₂-C₄-alkenyl, C₂-C₄-alkynyl, C₁-C₄ haloalkyl; whereinR^(a) is independently at each occurrence selected from: H, C₁-C₄ alkyland C₁-C₄ alkenyl.
 135. The method of claim 129, wherein the crosslinkeris or comprises N,N′-(1,2-dihydroxyethylene)bisacrylamide,N,N′-methylenebis(acrylamide), N,N′-ethylenebis(acrylamide), glycerol1,3-diglycerolate diacrylate, piperazine diacrylamide,N,N′-((ethane-1,2-diylbis(oxy))bis(ethane-2,1-diyl))diacrylamide,polyethyleneglycol diacrylamide (MW≤2000), 4-Arm PEG-Acrylamide(MW≤2000), N,N-bis(2-acrylamidoethyl)acrylamide, 1,2-dihydroxybis-acrylamide,N,N′-((ethane-1,2-diylbis(oxy))bis(ethane-2,1-diyl))diacrylamide or acombination thereof.
 136. The method of claim 129, wherein the polymerparticles are porous and/or have an average diameter of from 0.5 μm to10 μm, and/or wherein the coefficient of variation (CV) of the polymerparticles is less than 20%.
 137. The method of claim 129, wherein theparticles each absorb at least 20% of their weight of the aqueoussolution.
 138. A method of oligonucleotide sequencing, comprising:providing monodisperse cross-linked hydrogel polymer particles, whereineach particle comprises a polymer formed from (a) a hydrophilic vinylicmonomer having a log P_(oct/wat) (log P) of less than about 1, and (b) acrosslinker comprising at least two vinyl groups, wherein each particlecomprises one or more polynucleotides attached to the particle; andsequencing the polynucleotides by chemical field-effect transistor(chemFET) based sequencing or ion sensitive field-effect transistor(ISFET) based sequencing.
 139. The method of claim 138, wherein thecrosslinker comprises at least one compound of formula (IIa) or (IIb):

wherein R⁶ is selected from —C₁-C₆ alkyl-, —C₁-C₆ heteroalkyl-, —C₁-C₆cycloalkyl-, —C₁-C₆ hydroxyalkyl-, —C₁-C₆ ether-, or polyethercomprising 2 to 100 C₂-C₃ ether units; R⁷ and R⁸ are each independentlyselected from —H, —C₁-C₆ alkyl, —C₁-C₆ heteroalkyl, —C₃-C₆ cycloalkyl,—C₁-C₆ hydroxyalkyl, or —C₁-C₆ ether; R⁹ is —N(R¹¹)C(O)CH═CH₂; R¹⁰ isselected from —H and —N(R¹²)C(O)CH═CH₂; and R¹¹ and R¹² are eachindependently selected from —H, —C₁-C₆ alkyl, —C₁-C₆ heteroalkyl, —C₃-C₆cycloalkyl, —C₁-C₆ hydroxyalkyl, or —C₁-C₆ ether; optionally wherein oneor more of R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹ and R¹² are independentlysubstituted, where chemically possible, by 1 to 5 substituents which areeach independently at each occurrence selected from: oxo, ═NR^(a),═NOR^(a), halo, nitro, cyano, NR^(a)R^(a), NR^(a)S(O)₂R^(a),NR^(a)CONR^(a)R^(a), NR^(a)CO₂R^(a), OR^(a); SR^(a), S(O)R^(a),S(O)₂OR^(a), S(O)₂R^(a), S(O)₂NR^(a)R^(a), CO₂R^(a) C(O)R^(a),CONR^(a)R^(a), C₁-C₄-alkyl, C₂-C₄-alkenyl, C₂-C₄-alkynyl, C₁-C₄haloalkyl; wherein R^(a) is independently at each occurrence selectedfrom: H, C₁-C₄ alkyl and C₁-C₄ alkenyl.
 140. The method of claim 138,wherein the crosslinker is or comprisesN,N′-(1,2-dihydroxyethylene)bisacrylamide,N,N′-methylenebis(acrylamide), N,N′-ethylenebis(acrylamide), glycerol1,3-diglycerolate diacrylate, piperazine diacrylamide,N,N′-((ethane-1,2-diylbis(oxy))bis(ethane-2,1-diyl))diacrylamide,polyethyleneglycol diacrylamide (MW≤2000), 4-Arm PEG-Acrylamide(MW≤2000), N,N-bis(2-acrylamidoethyl)acrylamide, 1,2-dihydroxybis-acrylamide,N,N′-((ethane-1,2-diylbis(oxy))bis(ethane-2,1-diyl))diacrylamide or acombination thereof.
 141. The method of claim 138, wherein the particleseach absorb at least 20% of their weight of the aqueous solution. 142.The method of claim 138, further comprising sequencing thepolynucleotide by detecting a pH change in the local environment of thepolymeric particle with an ionic sensor.